Richard K. Wright

Hibernation alters the frog's immune system

The lymphomyeloid organs and blood leukocyte populations of the leopard frog, Rana pipiens, undergo conspicuous changes during hibernation at 4 degrees C. Within the blood, spleen, thymus, jugular bodies, and bone marrow there was a progressive loss of hemopoietic populations resulting in a marked lymphocyte depletion. Termination of the 135-day hibernation period resulted in the restoration of all hemopoietic elements in the blood and lymphomyeloid organs within 30 days. Frogs subjected to experimental hibernation and immunized showed weakened immune responses when brought from the hibernaculum. Plaque-forming cells (PFC) were lower in spleen, jugular bodies, and bone marrow, and serum antibody titers were also lower. Although the kinetics of the primary responses were essentially the same, the secondary responses differed suggesting major rearrangements with respect to the numbers of cells and their function in secreting antibody. The apparent lymphocyte aplasia may contribute to the absence of immunological responsiveness during periods of hibernation.

Temperature effects on ectotherm immune responses

Ectotherms are subjected to seasonal changes in temperature, a significant factor affecting their life processes and immune responses. Most teleost fishes, tropical anurans and reptiles can elicit immune responses over the entire range of their thermal tolerance limits. Their primary responses are temperature dependent with no apparent discrete temperature sensitive event. Temperature reductions from the species optima influence 1) response induction times, 2) levels of antibody produced, 3) times to reach peak antibody levels and to 4) reject allografts. Secondary or memory responses are temperature independent. These generalizations are also true for temperate amphibians and reptiles at non-hibernating temperatures. During hibernation however, primary and secondary responses cannot be elicited. This non-reactivity can be correlated with the decline in lymphocyte numbers and the thermal sensitivity of events occurring during the inductive and effector phases of immune responses.

Cellular Defense Systems of the Protochordata

Chordates are the largest phylum of deuterostomes and are divided into three subphyla: the Vertebra ta, Cephalochordata, and Urochordata. Although cephalochordates and urochordates lack a vertebral column, they do possess the three distinguishing characteristics of chordates at some time in their life cycle, i.e., a notochord, a dorsal tubular nerve cord, and pharyngeal clefts or gill slits. They are frequently referred to as the Protochordata, the subject of this chapter.

Leopard frog (Rana pipiens) spleen lymphocyte responses to plant lectins: kinetics and carbohydrate inhibition.

Mitogenic effects of the plant lectins phytohemagglutinin (PHA) and concanavalin A (Con A) on leopard frog (Rana pipiens) spleen lymphocytes in vitro were examined. At 22 ± 1°C, maximum stimulation in response to PHA and Con A occurs on day 4 with 1 pg PHA and 5–10 μg Con A. Lymphocyte stimulation can be inhibited by adding N-acetyl-D-galactosamine (NADG) to PHA cultures or α-methyl-D-mannopyranoside (αMM) to cultures containing Con A at time zero. Saccharide inhibition is concentration dependent: maximum inhibition is obtained with 75 mM NADG or αMM. Similarities between the kinetics of amphibian and mammalian mitogen stimulated lymphocytes and carbohydrate inhibition of lymphocyte activation suggest that certain biochemical characteristics of mitogen stimulation and cell surface receptors for mitogens have been phylogenetically conserved since amphibians first evolved during the Devonian period approximately 350 million years ago.

Crustacean defense strategies I. Molecular weight dependent clearance of dyes in the mud crab Scyllaserrata (Forskal) (Portunidae: Brachyura)

Clearance rates of dyes injected into the hemocoel of the mud crab, Scylla serrata, revealed several patterns of clearance and retention associated with physico-chemical and biological variables. Clearance rates remained constant: 1) when the molar concentrations of the injected dyes were equal, 2) after repeated injections of dyes and 3) after serum opsonization. Rates increased: 1) with higher molecular weight irrespective of charge, 2) at higher dye concentration and 3) at night concomitant with an increase in the hemocyte population. In contrast, clearance rates decreased with increasing crab size. Dye cleared from the hemolymph accumulated in the gills but not in the hepatopancreas, antennary glands nor gut. The retention of dyes in the gills increased with higher molecular weights. Granular hemocytes accumulated in the gill rachis and lamellae of dye treated but not in gills of normal and saline injected crabs. A role for hemocytes in molecular weight and concentration dependent clearance of dyes is suggested. Our findings are important for understanding the molecular basis of recognition in crustaceans.

Protochordate immunity—II. Diverse hemolymph lectins in the solitary tunicate Styela clava

Hemolymph lectins (agglutinins) of the tunicate Styela clava were analyzed by agglutination, cross-absorption and carbohydrate-hemagglutination inhibition using several vertebrate erythrocytes. Lectin activity was heat labile, dependent on divalent cations and refractory to neuraminidase-treated erythrocytes. Four lectins with different carbohydrate specificities were found. Carbohydrate specificities included L-rhamnose, D-glucuronolactone, maltose, D-galactosamine, D-mannosamine, D-galactose, hyaluronic acid and bacterial lipopolysaccharide. Since S. clava lectins can be inhibited by carbohydrates found in the extracellular capsule or cell wall of most bacteria, we propose that the lectins may be part of the tunicate immuno-defense system.

Fine structure of leopard frog (Rana pipiens) lymphocytes after in vitro transformation by phytohemagglutinin.

Abstract Stimulation of leopard frog ( Rana pipiens ) spleen lymphocytes in vitro with phytohemagglutinin (PHA) caused a gradual change from small or medium sized lymphocytes to lymphoblasts. Lymphocytes cultured with PHA formed cell clusters with uropods oriented towards the center, giving them a rosette-like appearance. Mitotic figures were observed within lymphocyte clusters. At the ultrastructural level, unstimulated lymphocytes had sparse cytoplasm containing a few mitochondria, scattered free ribosomes and nuclei containing heavy clumps of heterochromatin. In contrast, after PHA stimulation, lymphocytes possessed the typical ultrastructure of lymphoblasts. The nucleus contained loosely packed euchromatin and prominent nucleoli. Ribosomes were aggregated to form polysomes, rough endoplasmic reticulum was sparse and mitochondria numerous. Uropods contained a well developed Golgi apparatus, centrioles with associated microtubules, various membrane bound bodies and abundant mitochondria and polysomes. The fine structural features of transformed frog lymphocytes are similar to those observed in several mammalian species, suggesting that this facet of lymphocyte biology has been conserved during evolution.

Lymphoid Organs and Amphibian Immunity

Immunological responses are physiological mechanisms which endow an animal with the capacity to recognize foreign materials and to eliminate them. In order to execute the functions of immunity, a ubiquitous cell system has evolved within the vertebrates known as the lymphoreticular system. This collection of cellular elements is distributed strategically throughout the body and housed primarily in various lymphoid organs. Observations on the morphological development of the vertebrate lymphoid system (1) reveals that as one examines the phylogenetic scale from agnathans to birds and mammals, the number, location and diversity of lymphoid organs increases. By analyzing the lymphoid system of primitive vertebrates, one can thus gain answers as to how the immune system of advanced vertebrates evolved.

Activation of leopard frog (Rana pipiens) spleen lymphocytes by concanavalin A and phytohemagglutinin

Abstract Activation times for concanavalin A (Con A)- and phytohemagglutinin (PHA)-induced lymphocyte mitogenesis in leopard frogs were determined by competitive inhibition analysis using α-methyl- d -mannopyranoside and N -acetyl- d -galactosamine. Con A activation required approximately 18–20 hr whereas PHA was required for at least the first 24 hr. Temporal events for mammalian lymphocytes are similar suggesting that activation parameters of lymphocyte biology have remained unchanged since the first vertebrate tetrapods evolved.

Bone marrow reconstitution of immune responses following irradiation in the leopard frog, Ranapipiens

The bone marrow of Rana is an important source of cells capable of maintaining individual viability, responding to Concanavalin A (Con A) and producing PFC against sheep erythrocyte (SRBC) antigens. Frog marrow is more effective than the spleen in maintaining life. Radiation destroys the ability of frogs to respond to SRBC immunization (lack of bone marrow and spleen PFC, serum antibody) and bone marrow/spleen cells to respond to Con A, i.e., bone marrow and spleen contain radiation-sensitive cells. Shielding one hind leg during irradiation leads to reconstitution of bone marrow/spleen PFC responses, antibody synthesis and individual viability. Our results suggest that bone marrow is: a) the source of stem cells, and b) the source of mature T- and B- lymphocytes that can recirculate within the immune system.