Reactions in vivo and in vitro produced by a soluble substance associated with delayed-type hypersensitivity.

The cellular basis for delayed-type hypersensitivity was established by the passive transfer of delayed-type reactions to normal guinea pigs by means of lymphoid cells obtained from hypersensitized animals.' Attempts to demonstrate a mediator of delayed-type hypersensitivity in sera or lymphoid cell extracts from sensitized individuals have been unsuccessful in experimental animals.2 Recently a technique 3' 4 for studying this immunological response in vitro has been developed which is based on the observation' that migration of peritoneal exudate cells from sensitized guinea pigs is inhibited by the presence of specific antigen. In this system, it has been found that the immunological information for this response is possessed by the lymphocytes, while the macrophages serve as indicator cells which migrate.6 When such sensitized lymphocytes are cultured in the presence of specific antigen, they elaborate into the medium a substance, probably a protein, capable of inhibiting the migration in vitro of normal macrophages.6-'0 This substance, termed migration inhibitory factor (MIF), is produced by lymphocytes from guinea pigs exhibiting delayedtype hypersensitivity but not from animals producing only circulating antibodies,3' 6 and is produced only after the sensitized lymphocytes interact with the specific antigen.1' This report will describe (1) the production of MIF by sensitized lymphocytes cultured in serum-free media, (2) partial purification of the active material, and (3) the production of skin reactions in normal guinea pigs by such purified preparations. Materials and Methods.-Animals: Random-bred Hartley guinea pigs were purchased from commercial breeders, and inbred strain XIII guinea pigs were obtained from our own breeding colony. Sensitization: Guinea pigs were sensitized to tuberculin by the injection of complete Freund's adjuvant (3.3 mg/ml H37Ra mycobacteria, Difco) on one occasion as follows: 0.1 ml into each footpad and 0.6 ml into the nuchal muscle. Three to 6 weeks later, each animal was tested intradermally with 10 gg of tuberculin purified protein derivative (PPD, obtained from the Ministry of Agriculture, Fisheries and Food, Central Veterinary Laboratory, Weybridge, England). Animals showing reactions of 15 mm or greater were used as cell donors. Tissue cultures of lymphocytes: Lymphocytes were obtained from brachial, axillary, cervical, and inguinal lymph nodes, which were teased apart with stainless-steel rakes2 in Hank's solution containing 10% normal guinea pig serum (NGPS, prepared from blood obtained by cardiac puncture). The cells were washed twice in Hank's solution by centrifugation at 200 X g and finally suspended in Eagle's minimum essential medium (MEM) containing 100 U penicillin/ml, and 100 Ag streptomycin/ml. Finally, the cell density was adjusted to 1.2 X 107 cells/ml, and the cells were cultured in plastic T-flasks (Falcon Plastic Co.) for 24 hr with PPD, 25 jsg/ml. No serum was added. Afterward,