Enhancement of in Vivo Incorporation of Labeled Precursors into Dna and Total Protein of Mouse Lymph Nodes after Administration of Thymic Extracts.

In a study of the protein composition of lymphoid tissue, Roberts and White1 in 1949 reported that a fraction isolated from calf thymus produced thymic hypertrophy when injected for a ten-day period into adult rats. Since this suggestion of a humoral role for the thymus in stimulating lymphocytopoiesis, considerable additional evidence'has appeared. This effect appears to have some relation to the role of the thymus in the acquisition of immunological competence after birth6 or after whole body irradiation.' Investigation into the apparent lymphocytopoietic activity of thymic extracts has been hampered by the lack of a rapid assay method. Utilization of a peripheral blood lymphocytosis as assay of lymphocytopoiesis is not only tedious, but is subject to criticism on theoretical grounds.8 Estimation of the rate of lymphocyte proliferation by measurement of incorporation of isotopically labeled precursors appears to offer many advantages in the detection of lymphocytopoietic activity in various extracts of thymus glands. Previous work in this laboratory9 has shown the usefulness of this approach in studying the effects of steroid hormones on lymphoid tissue. In this communication we wish to report the enhanced in vivo incorporation of tritiated thymidine into DNA, and of glycine-2-C'4 into total protein of mouse lymph nodes following injection of either calf, rat, or mouse thymic extracts. The data obtained utilizing labeled thymidine afford a basis for assessing the activity of thymic extracts in promoting lymphocyte proliferation. Materials and Methods.-Frozen calf thymus, obtained from a local abattoir, fresh rat thymus, obtained from 180-240-gm male Sprague-Dawley rats, or thymus from groups of adult, CBA mice, was homogenized in 0.15 M NaCI or in 0.1 M phosphate-Versene buffer, pH 5.7 (tissue: solvent = 3: 1) at 0-40C. The homogenate was then centrifuged at 700 X q in an International refrigerated centrifuge at 0C for 15 min and the supernatant then centrifuged at 105,000 X g in a Spinco model L ultracentrifuge for 1 hr. The clear supernatant obtained was used for assay. Some thymic extracts were prepared according to the method of De Somer and co-workers4 by incubating minced thymus aseptically in Hank's solution for 48 hr with subsequent separation of the cellfree supernatant. All extracts were used immediately after preparation or stored at -20'C for up to three weeks. Extracts of rat spleen and lymph nodes were prepared with 0.1 M phosphate buffer as with thymus above. Inbred male CBA mice, 55-70 days old, from a colony maintained in our laboratory were used for all assay experiments. Mice born on the same or on successive days were used together to make the experimental groups as homogeneous as possible. Extracts were injected intraperitoneally into groups of animals at periods of 24-72 hr before sacrifice. Control groups were injected with buffer. Two hours before sacrifice, a pulse label of H3-thymidine (3 mc/mmole, Schwarz BioResearch, New York), 10 or 15 sc per mouse, or glycine2-C14 (12 mc/mmole, New England Nuclear Corp., Boston), 0.20 ic/gm body weight, was injected intraperitoneally. The mice were sacrificed by cervical fracture. Axillary and inguinal lymph nodes, thymii, and spleens were removed rapidly from each group of mice, weights of the respective pooled tissues recorded, and then placed in 0.15 M saline at 0-40C. In the experiments utilizing H3-thymidine, the lymphoid organs were homogenized at 0-4oC