C. R. Jansen

LYMPHOCYTE PRODUCTION MEASURED BY EXTRACORPOREAL IRRADIATION, CANNULATION, AND LABELING TECHNIQUES*

The lymphatic system has, truly an ancient history that is ably reviewed in Yoffey and Courtice.’ Apparently lymphatics were first observed around 300 B. C. In the 1600s, mesenteric lymphatic vessels and the thoracic duct were described. The classical injection of lymphatic vessels throughout the body by the Hunter Brothers in the 1700s are common knowledge. In 1800, Ludwig commenced cannulation of different lymphatic vessels. In 1851, lymphocytes were first seen within the lymph by Virchow.2 In 1875, Fleming observed mitoses in the germinal centers of lymph nodes. In the late 1800s, Ernest Starling pointed out that lyrnphatic vessels serve as a mechanism by which protein lost from the blood vessels can be returned to the blood. He thus firmly established one major function of the lymphatic system. In this century, a vast literature has developed on the lyrnphopoietic system that is too unwieldly to review in this brief paper. In Yoffey and Courtice’s book on lymphoid tissue, a large mass of the current literature is analyzed. A variation in the concentration of lymphocytes in afferent and efferent lymph has been presented by E h r i ~ h . ~ Following prolonged antigenic stimulus, the output of lymphocytes in the efferent lymphatic ducts of lymph nodes was greatly increased. There is also an increase in the lymphocytes in the lymph while passing through a lymph node. Drinker and Yoffey4 point out that this can not be explained by reabsorption of water in the node because the protein content of peripheal and central lymph is identical in amount. In fact, “if the blood capillaries of the lymph nodes are capable of large scale absorption of protein, they are not only unique members of the blood capillary system, but at the same time display a miraculous ability to absorb proteins so equally as to make afferent and efferent lymph precisely equal in protein content.” Much valuable information had been obtained by cannulation techniques. However, it was with the development of radioactive labels of DNA to tag the new cell production that knowledge on the magnitude of cell production and lifespan of cells began to grow. Again the literature is too large to review in detail. However, the classical studies of O ~ g o o d , ~ Otteson,6 and Hamilton’ are pertinent. Hamilton, utilizing C14-labeled adenine and guanine precursors of DNA, extended Otteson’s ideas on the possibility of two types of lymphocytes ( 1 ) one cell with a long lifespan and (2) another cell with a short lifespan. Alternatively, he could explain his data on the basis that there may be significant reutilization of the labeled DNA or its fragments. Hamilton also first pondered the possible significance of DNA reutilization in perpetuating immune responses. Gowans et ~ l . , * . ~ in a series of precise studies, has pointed out by utilizing classical cannulation techniques, radioactive labeling, and autoradiography that there is a significant recirculation of lymphocytes from blood to the lymphocytic organs and back to the blood again. His group has also confirmed the earlier

Studies on lymphocytes: V. Short in vivo DNA synthesis and generation time of lymphoid cells in the calf thoracic duct after simulated or effective extracorporeal irradiation of circulating blood☆

Abstract The proliferative pattern of lymphoid cells in the thoracic duct of the calf was studied by the use of tritiated thymidine and autoradiographic evaluation of lymph samples taken at frequent time intervals. The fact that the thoracic duct contains a considerable number of cells in mitosis makes it possible to define more precisely the time parameters of their generation cycle in one and the same animal. A combined analysis of the mitotic labeling index (MLI) and the mean grain count per labeled mitotic figure (MGC/LMF), both as a function of time after a single i.v. injection of 3H-thymidine, revealed an in vivo G2 period of 30 to 40 min, a DNA synthesis time of 3 1 2 hr and a generation time of 5 1 2 to 6 hr for the majority of cells. The duration of these separate phases of the cell cycle was not changed to any appreciable degree by 48 hr of extracorporeal irradiation of the circulating blood (ECIB) prior to the study of lymphoid cell proliferation.

LYMPHOID CELL LINES IN THE THORACIC DUCT OF THE CALF WITH DIFFERENT GENERATION TIMES.

Small lymphocytes represent a mixed population with varying life spans (Ottesen, 1964; Everett et al., 1964; Robinson et al., 1965). In the present report evidence will be presented indicating that at least two populations of lymphoid cells with different tinctorial properties and unequal DNA-synthesis and generation times exist in the thoracic duct of calves. The data are based on a combined analysis of the mitotic labeling index (MLI) and the mean grain count per labeled mitotic figure (MGC/LMF), both as a function of time after a single intravenous injection of Thymidine-3H. This method was previously found to necessitate the least number of assumptions in evaluating proliferative patterns of cell lines without detectable cytoplasmic differentiation (Janett et al., 1966).