After immunization of carp with sheep red blood cells, the spleen accounts for only 5% of the total number of plaque-forming cells (PFC). In addition, thymus, peripheral blood and heart contained low numbers of PFC (< 0.5, 1 and 0.5%, respectively). Pronephros and mesonephros were the major antibody-forming organs (53 and 40% of total PFC, respectively). The temperature dependence of the antibody-forming cell response in spleen, pronephros and mesonephros as studied in animals kept at 12-24 degrees. Lowering temperatures induced a delay in the peak of the primary response but had no effect on the magnitude of the response. The temperature-peak day relationship indicated that there are steps in the primary immune response of carp differing in temperature sensitivity. The anamnestic character of the secondary response was clearly demonstrated at 24 and 20 degrees but lost at 18 degrees.

The immune system of cyprinid fish. Kinetics and temperature dependence of antibody-producing cells in carp (Cyprinus carpio).

The primary immune response of the toad, Bufo marinus, to Salmonella adelaide flagella has been analysed in terms of three immunological parameters: (a) the kinetics of appearance and morphology of cells producing specific antibodies; (b) antigen retention in one of the lymphoid organs, the jugular bodies; and (c) the nature of the immunoglobulins produced. It was found that toads responded to the bacterial antigen by producing antibody-forming cells and serum antibody in quantities comparable to those observed in mammals. In the early phase of antibody production, small and medium lymphocytes played an important role in antibody synthesis. In the later stages of the response, large cells which resembled immature plasma cells were the predominant producers of antibody. Studies on the incorporation of tritiated thymidine suggested that the majority of antibody-forming cells observed during the logarithmic phase of the response arose by division. Electron microscopic studies showed that the localization of radioactively-labelled antigen in the jugular bodies was similar to the follicular localization in mammalian lymph nodes, i.e. the antigen was associated with the surfaces of cells, rather than with intracellular sites. Although the toad was found to possess distinct classes of 18S and 7S immunoglobulins, antibody activity to this antigen was present only in the 18S molecules.

These results are consistent with the hypothesis that the major patterns of differentiation and proliferation of immunologically competent cells, antigen retention and immunoglobulin structure have emerged by the phylogenetic level of anuran amphibians.

Cellular and humoral aspects of the primary immune response of the toad, Bufo marinus.

The reticuloendothelial system of Aschoff comprises those phagocytic cells which take up carbon and dyes after these have been introduced into the animal (Clark, 1965). Both morphologically and functionally, this system is so intimately related to lymphoid cells and to the immune response that it would be unrealistic to consider one without the other. Furthermore, in amphibians, myeloid and lymphoid tissue occur along with macrophages and other reticuloendothelial cells, in close relationship within the same organs, the spatial separation between myelopoiesis and lymphopoiesis being less distinct in poikilotherms than in mammals (Yoffey, 1960). The organs concerned with these functions in the Amphibia are listed in Table 1. In this chapter we shall consider the structure and function of these organs.

RES Structure and Function of the Amphibia

SYNOPSIS. Lymphoid tissue and lymphopoiesis in the urodeles and anurans was reviewed. Current information suggests the spleen as the principal site of lymphopoiesis in urodeles. Anurans present a more complex situation and the principally spleenic reaction of Rana pipiens to “red-leg” organisms was contrasted with the kidney intertubular lymphoid reaction of Bufo marinus to bovine serum albumin. Intracisternal accumulations of slightly electron dense material, probably immunoglobulins, were more frequently found in Bufo marinus plasma cells than cells with undistended cisternae suggesting some specialization of these cells in cold-bloodedvertebrates. The superficial nature of our current knowledge of lymphoid tissue function and immunity in this groups was emphasized.

Lymphopoietic Tissue and Plasma Cells in Amphibians

Antibody-forming cells (AFC), developing in toad spleen after stimulation with polymerized flagellin, were studied with an immune adherence assay. Differentiation was followed by several parameters: thymidine uptake to monitor dividing cells; equilibrium density centrifugation in albumin gradients to monitor cell density; microscopic measurements and sedimentation velocity separation to monitor cell size; stained preparations to follow cell morphology. Almost all AFC observed early in the response were dividing cells; the proportion of dividing AFC dropped to 4% 2 wk after stimulation. The earliest AFC detected (3 days) formed a relatively homogeneous light density population, and were purified 17-fold by equilibrium density centrifugation. As the response developed, additional denser peaks were found, so that late in the response dense AFC predominated. Dividing AFC were confined to the light density region throughout the response. Cell diameter measurements revealed that the earliest AFC were all very large cells. In a manner analogous to the density changes, smaller AFC appeared as the response developed until they finally comprised the majority of the AFC population. Dividing AFC were always relatively large, but encompassed a wide range of sizes. Sedimentation velocity separation was employed in a closer study of the immature AFC; they were purified 140-fold by this procedure. The earliest AFC consisted of several readily separable size populations in the range 9–18 µ diameter. The presence of separate peaks related by factors of two in volume suggested that the largest cells undergo a series of halving divisions before entering a division growth cycle. The results suggest an AFC differentiation sequence from a very large, light density, dividing "blast" cell to a nondividing cell with the size, density, and morphological appearance of a small lymphocyte. Stages of this sequence can be defined and selected out for investigation, using sedimentation velocity and equilibrium density centrifugation as complementary techniques.

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DIFFERENTIATION OF ANTIBODY-FORMING CELLS IN TOAD SPLEEN : A Study Using Density and Sedimentation Velocity Cell Separation

Cells with the light microscopic and ultrastructural features of mamalian plasma cells were observed in lympoid tissues of long-term stimulated Bufo marinus, the marine toad. Cells with this morphology were the only cells which could be stained by the fluorescent antibody method. The cellular basis of immunoglobulin formation in cold-blooded vertebrates thus appears to be the same as that in mammals. Bufo marinus plasma cells were most abundant in the kidney lymphoid tissue, and in the margins of granulomas formed at the sites of injection of antigen in complete adjuvant.

Dyer Rf

Papers

Amphibian plasma cells.