A technique for collecting lymph for long periods of time from the hind limbs of conscious sheep is described. Spontaneously flowing lymphatic fistulhe have been established in fifteen sheep and the number and type of the cells coming from the popliteal lymph node have been studied. Following the operation the output of cells in the lymph was severely depressed for a period of about 24 hr. Under basal conditions the rate of lymph flow varied between 2 and 10 ml./hr. and the output of cells between 30 and 100 millions/hr. When the popliteal node was stimulated by the subcutaneous injection of human serum globulin into the lower leg, the cell output increased and different types of cell appeared in the lymph. Large, primitive transitional cells, and cells of the plasma series were present in considerable numbers during the response. Some observations on the cell content of peripheral lymph, collected from chronic afferent lymphatic fistuloe, are also presented. The significance of the cell types seen in the lymph collected during these experiments is discussed.A DETAILED study of the number and type of cells coming from a single lymph node might provide useful information about the origin, fate and function of lymphocytes and associated cells. To obtain satisfactory data on these subjects, it is necessary to collect lymph coming from a defined area of drainage for long periods of time under physiological conditions. Experiments based on the cannulation of major lymphatic trunks cannot yield direct evidence concerning the output of cells from individual nodes and the results obtained with this type of experiment may be confounded by metabolic disturbances caused by the loss of large quantities of cells, fluid, electrolytes and protein from the body.Most previous attempts to collect efferent lymph from peripheral lymph nodes have been carried out acutely in anaesthetized animals [Menkin and

The popliteal nodes of sheep were perfused continuously for over 100 hours with a solution containing 3H-thymidine. The labelling pattern of the lymphocytes in the efferent lymph from the perfused nodes showed that under normal conditions not more than 4 per cent of these lymphocytes were actually produced in the node. The transitional cells, large lymphocytes and cells of the plasma cell series which appeared in the lymph following antigenic stimulation of the node were all produced in the node.

No abstract

The changes in the cell population in the efferent lymph from the popliteal node have been studied in unanaesthetized sheep given subcutaneous injections of various antigens into the lower part of the leg. The antibody content of the cells and of the lymph plasma has been measured during the immune responses which followed the injection of the antigens.Human serum globulin, chicken red cells and killed Salmonella typhi were the antigens used. The chicken red cells and Salmonella organisms were virtually all retained in the lymph node whereas human serum globulin was mostly recovered in the efferent lymph. With all the antigens, a characteristic cellular response occurred in the efferent lymph. Initially there was an increase in the 'output of mature lymphocytes followed by the appearance of primitive stem cells and finally plasma cells. Antibody was detected in the cells and lymph plasma in the experiments with particulate antigens and in the lymph in experiments with human serum globulin. When a second dose of particulate antigen was injected, the response was always shorter and more vigorous than after the first. There was little difference between the primary and secondary responses after human serum globulin.The cell population of afferent lymph was found to change in roughly the same way as that of efferent lymph when an antigen was injected into the leg. All the cell types which characterized the immune response in the efferent lymph were identified in the afferent lymph.

The large amounts of IgA made by plasma cells in the gut and mesenteric nodes produce high levels of IgA in the intestinal and thoracic duct lymph which are difficult to reconcile with the low levels found in the blood of most species.In rats, blood serum levels of 0.05-0.18 mg/ml have been reported while mesenteric or thoracic duct lymph with about one-third of the total immunoglobulin level of blood contain 0.6 mg/ml (1-5). Assuming a flow rate of 1-3 ml/ h for thoracic duct lymph of rats and a blood plasma vol of 8 ml the amount of IgA which daily enters the blood from the thoracic duct is 20-50 times greater than the circulating plasma pool. The evident disappearance of this IgA from the blood suggests that it is either catabolized Very fast or exported. The availability of IgA from rat plasmacytomas (6) now makes it possible to purify enough IgA to explore these possibilities, Preliminary experiments had shown (a) that IgA disappeared from the blood very much faster than similarly labeled IgG2; (b) that feces collected during the day of injection contained antigenically intact radiolabeled IgA; and (c) that in accord with Lemaitre-Coelho et al. (7) rat bile had IgA levels some 10 times higher, and IgG levels 30-50 times lower, than those of blood serum. As the liver of healthy mammals contains no cells considered capable of immunoglobulin synthesis, it seemed likely that the biliary IgA was derived from the blood by active transport. Materials and MethodsImmunoglobulins and Antisera. IgA was isolated from the ascitic fluid of Lou/Wsl rats bearing the IR461 plasmacytoma. After salt precipitation the material was fractionated twice on a 2.6 × 100 cm column of Ultrogel Ac22. The IgA formed by IR461 is heterogeneous with regard to size, and, like normal rat IgA, mainly oligomeric (5). The fraction least contaminated with other proteins (Fig. 1) had a sedimentation rate of 13.2 S; it contained a-macroglobulin detectable in immunoelectrophoresis and estimated as 0.3 mg/ml. Other contaminants account for less than 5% of the total protein (5.4 mg/ml) so the fraction was about 90% IgA.IgG2 was made from serum of Lou/Wsl rats bearing the IR33 plasmacytoma which secretes IgG2a. The material used in the clearance studies, eluted from DEAE cellulose with 0.01 M phosphate buffer pH 7.4, contained some IgG2b but no IgG1.Antisera obtained by immunizing rabbits with purified myeloma IgA contained anti-idiotypic and other unwanted antibodies, most of which were absorbed out with a pseudoglobulin