Amphibian lymphokines: 2. Migration inhibition factor produced by antigenic and mitogenic stimulation of amphibian leucocytes.

Migration of Rana temporaria peritoneal exudate cells (PEC) was examined in vitro using both direct and indirect assay systems. After sensitization in vivo followed by in vitro challenge 7-21 days later with the appropriate sensitizing antigen, spleen cell culture supernatants were obtained which inhibited the normal in vitro migration of PEC from non-sensitized animals. Cultures of spleen cells with mitogen also gave rise to supernatants with migration inhibitory properties. Sephadex separation of supernatants showed that maximum inhibitory activity was present in the 27-50,000 MW range and, furthermore, that this inhibition was blocked by alpha-L-fucose, but not by beta-D-galactose. The inhibition did not appear to be species specific. The results indicate that following appropriate stimulation amphibian leucocytes produce a soluble, migration inhibition factor (MIF) with characteristics similar to those described for the mammalian lymphokine MIF.

Amphibian lymphokines: 1. Leucocyte chemotactic factors produced by amphibian spleen cells following antigenic and mitogenic challenge in vitro.

The production of soluble chemotactic factors by Rana temporaria leucocytes following in vitro stimulation with antigen or mitogen was examined. Stimulated spleen cells were found to produce a soluble factor with a molecular weight (MW) of between 16,000-27,000 daltons which was chemotactic for Rana peritoneal cells. Rana peritoneal cells also showed directional movement towards solutions of casein, which is a potent chemo-attractant for mammalian lymphoid cells.

Changes in plasma levels of calcium and in bone marrow mitosis after antigenic challenge in rats and mice.

When rats or mice were immunized with sheep red blood cells, bacterial lipopolysaccharides or bovine serum albumin, a proliferative response could be detected in the bone marrow and spleen. This response was associated with a hypercalcaemic phase. Parathyroidectomy, which resulted in a protracted hypocalcaemia, prevented the development of an increase in levels of plasma calcium. This operation also prevented the rise in bone marrow proliferations following antigenic challenge, but did not ablate the normal proliferative response to antigen by cells in the spleen. Antibody production and numbers of antibody-forming cells were not significantly reduced by parathyroidectomy. These results suggest that there is a pool of antigen-insensitive cells in the bone marrow which are stimulated after antigenic challenge. It is postulated that these events were mediated by the development of a parathyroid-dependent hypercalcaemia which stimulates the cells non-specifically. These events may form part of a cellular homeostasis, replacing cells in peripheral lymphoid tissues.

Electron microscopic studies of the developing amphibian thymus.

Electron-microscopic studies of larval Xenopus laevis thymus at three different stages of development are presented. Studies of thymus from 5 and 8 day old larvae (ages when early thymectomy is performed) reveal a relatively undifferentiated rudiment composed of epithelial cells and lymphoid cell precursors. These findings are in marked contrast to ultrastructural observations of 30 day old larval thymus, where there exists a well defined corticomedullary differentiation with an abundance of small lymphocytes in the cortical region.

Splenic involvement in amphibian transplantation immunity.

The spleen has been identified as a centre of alloimmune reactivity in control Xenopus. Levels of tritiated thymidine labelling and pyroninophilic cells are elevated in spleens of skin-allografted toadlets when compared with autografted and nongrafted animals. Second-set alloimmune reactivity can be transferred by implanting a spleen from a donor that has rejected one or two grafts into a nonsensitized host. Spleen donor and host in these experiments were mutually tolerant, following reciprocal transfer of embryonic tissue grafts. In contrast, studies on the uptake of tritiated thymidine and levels of pyroninophilia in animals thymectomized at 7 or 8 days of age suggest lack of splenic involvement in the chronic first-set allograft rejection that can still occur in the absence of the thymus. The lymphoid organ origin of "thymic-independent" alloimmunity still awaits clarification.

Critical role of the thymus in establishing humoral immunity in amphibians: studies on Xenopus thymectomized in larval and adult life.

Proliferative studies on spleen lymphocytes suggest the absnece of induced cellular activity to sheep erythrocytes in Xenopus thymectomized at 7 days of age. Immunological studies (measurement of splenic rosette-forming and plaque-forming cells and serum haemolysins) show that throughout larval life the thymus plays a critical role in establishing reactivity to red cells, since thymectomy as late as 40 days (stage 57) impairs the antibody response. In contrast, thymectomy during metamorphosis or in early adult life has no apparent debilitating effect.

Allograft rejection in larval and adult Xenopus following early thymectomy.

The effect of thymectomy (performed on 7- to 8-day old larvae) on the development of alloimmunity in the clawed toad is examined. Rejection of first-set skin grafts applied in larval life (30 days of age) and at intervals following metamorphosis (70 to 370 days of age) was always impaired following thymectomy. However, most of the thymectomized animals, even those grafted as larvae and blocked in the larval state by thiourea treatment, were able to destroy the transplants, albeit with prolonged rejection times. The pattern of graft rejection in thymectomized larvae was similar to that seen in adults. After thymectomy, these seemed to be no improvement or impairment of the alloimmune response with the passage of time. Experiments with second-set grafts applied subsequent to first-set destruction demonstrated the existence of an alloimmune memory component throughout ontogeny in both control and thymectomized animals. The results indicate that a population of cells involved in graft rejection develops early in ontogeny, independent of the thymus and the emergency of bone marrow at metamorphosis.

The effect of thymectomy at different stages of larval development on the immune response of the clawed toad to sheep erythrocytes.

The role of the thymus during the first three weeks of larval life in the maturation of humoral immunity in the amphibian, Xenopus laevis, is examined. Thymectomy throughout this period dramatically affects the heterologous red cell response of young adults. Sheep erythrocyte (SRBC) administration elicited haemolytic antibody production in the blood and in the spleen (measured by the appearance of plaque forming cells) of control animals, but failed to do so in all thymectomized toadlets. Moreover, use of the immunocyto-adherence assay, which proved to be a sensitive test for SRBC reactivity in control Xenopus, indicated a complete absence of induced responsiveness to this antigen in the spleens of thymectomized toadlets, even in those animals thymectomized as late as 22 days of age. In contrast to allograft immunity in Xenopus, Which is suppressed only by thymic ablation during the first two weeks of larval life, the development of reactivity to heterologous erythrocytes therefore requires an additional period of thymic influence during ontogeny. These experiments support a concept of thymus-dependent cell heterogeneity in amphibians.