REGULATION OF THE SECONDARY ANTIBODY RESPONSE IN VITRO

Abstract

Two opposite effects of actinomycin D on antibody synthesis have been studied in organ cultures of rabbit lymph node fragments. These cultures were prepared from previously primed rabbits and stimulated with antigen(s) on day 0 to yield a secondary response, whose inductive phase extended to about day 9 and whose productive phase may last for several months in the serum-free medium described here. Concentrations of actinomycin D above 0.01 µM (0.012 µg/ml) produce inhibition of antibody synthesis during both phases of the response. However, antibody synthesis is about 10 times more sensitive to inhibition by this drug when it is added during the inductive phase than during the productive phase. During the latter phase, synthesis is more rapidly terminated as the drug level approaches 10 /µM (12.5 µg/ml). At this level the 50% synthesis time is about 2.8 hr, which is identical with that found when 5–10 µM puromycin is added to the medium of parallel cultures. Transient enhancement of antibody synthesis is frequently produced by a brief exposure to low levels of actinomycin D (generally less than 0.01 µM). Enhancement appears in precise temporal association with actinomycin pulses added for 2 days or less only between days 6 and 16. This apparent enhancement of antibody synthesis resembles the increased enzyme synthesis described by Garren et al. (6) and led to a search for an antibody-inhibitory material (AIM) whose synthesis might be stopped preferentially by low levels of the drug. Stimulated lymph node cultures produce between days 6 and 15 a nondialyzable material which inhibits antibody synthesis during the productive phase of heterologous antigen-antibody culture systems. Just as enhancement with low levels of actinomycin D appears within 2 hr after the drug has been added to cultures, so inhibition occurs within 4 hr of adding AIM to cultures during their productive phase. These observations suggest that AIM is analogous to the translational "repressor" postulated by Garren et al. (6). AIM has relevance in two areas of immunology: (a) it may be the explanation for many examples of antigenic competition, and (b) it may represent a normal control mechanism for the productive phase.