Interaction between 'sensitized lymphocytes' and antigen in vitro. I. The release of a skin reactive factor.

Peritoneal exudate lymphocytes (PEL), purified on glass beads and lymph node (LN) cells from guinea-pigs immunized with tubercle bacilli were cultured for 24 hours, in serum-free medium, without and with various concentrations of Tuberculin PPD. Supernatants obtained from cultures with 10 μg PPD/10 lymphocytes provoked an intense inflammatory reaction, when injected into the skin of normal guinea-pigs. PEL were more active than LN cells from the same animals. The reaction was characterized by erythema and induration, with a peak between 3 and 6 hours and histologically a mixed polymorphonuclear—mononuclear infiltrate in the dermis was seen. When fractionated on Sephadex G-200, skin activity of both PEL and LN supernatants was concentrated in a peak corresponding to the molecular weight of serum albumin, while in LN material some activity was also present in a small molecular weight peak. The active material could be separated from albumin by chromatography on DEAE-cellulose. Immunoelectrophoretic analysis of skin reactive peaks detected a slow α-globulin in both PEL and LN supernatants. PPD in the form of a complex with a protein precipitated by anti-γG antiserum, was detected in the skin-active Sephadex peak III of LN supernatants, by radioimmunoelectrophoresis. Skin activity was precipitated with ammonium sulphate at 66 per cent saturation and was destroyed by pepsin treatment. Formation of the skin-active material was depressed by Puromycin and Actinomycin-D and the development of skin inflammation was suppressed by pretreatment of the recipient with anti-lymph node extract serum. Evidence for antigen induced specific synthesis and release of an α-globulin in PEL and LN cultures was found but its relation to the skin active material is unknown.