Rachel O. Johnson

Apoptosis in thymus of adult Xenopus laevis.

Thymocyte apoptosis in adult Xenopus laevis is demonstrated on agarose gels and is quantified by propidium iodide incorporation using flow cytometry. Basal apoptotic levels are increased after in vitro exposure to a glucocorticoid, dexamethasone (DEX), and to the lectin, phytohemagglutinin (PHA). To determine the role that newly introduced antigenic determinants may play in this regard, a repertoire of altered-self antigens was created by exposing thymuses in vitro to trinitrobenzene sulfonic acid (TNBS) thereby derivatizing self-cells and proteins via 2,4,6-trinitrophenyl-acetic acid conjugation. An increase in apoptosis in TNBS-treated thymuses is observed. Thus, the thymocytes of adult Xenopus laevis are susceptible to apoptosis when induced by a glucocorticoid, a lectin, and by altered self, antigen activation.

Expression of a Fas-like proapoptotic molecule on the lymphocytes of Xenopus laevis.

Ligation of the externally expressed Fas (APO1/CD95) molecule will initiate programmed cell death (apoptosis), in many mammalian developing and adult cells. Fas-induced apoptosis has not been demonstrated with the cells of any non-mammalian vertebrate. We immunostained suspensions of splenocytes from adult Xenopus laevis, the South African clawed toad, with a polyclonal rabbit anti-human Fas antibody raised against the amino acid residues 321-335 of human Fas. The binding was specific, as it was dramatically reduced by preincubation of the antibody with the Fas peptide used to make it, but not with a Fas-ligand (FasL) peptide. The binding was enhanced after in vitro exposure of the splenocytes to phytahemagglutinin (PHA), a T cell mitogen and apoptogen in this species. Sections of developing Xenopus larval tissue were also immunostained with the polyclonal rabbit anti-human Fas antibody. Consistent binding of thymocytes and splenocytes was not observed until early metamorphosis in these immunological sites. A monoclonal mouse anti-human Fas antibody, previously used to stimulate apoptosis in mammalian cells, induced significant levels of apoptosis in adult Xenopus splenocytes and additionally, bound specifically to a splenocyte extract, as assayed by ELISA. Thus, a molecule on Xenopus splenocytes shares both structural and functional homologies with human Fas, indicating the evolutionary conservation within vertebrates of this means of initiating apoptosis.

Apoptosis in the thymus of developing Xenopus laevis

Metamorphosis in Xenopus laevis is a time when thyroxine and glucocorticoid levels rise, dramatic morphological and physiological changes take place, and tolerance is established to newly expressed adult antigens. In vitro exposure of thymocytes tested at different metamorphic stages, to the T-cell lectin, phytohemagglutinin (PHA), stimulates increased apoptosis, but incubation with the synthetic glucocorticoid, dexamethasone (DEX), fails in this regard. Altered-self antigenicity, following trinitrobenzene sulfonic acid (TNBS) treatment, increases apoptosis only in the late stages of metamorphosis. Developmentally blocked metamorphosing larvae demonstrate low thymic apoptotic rates that are also unaffected by in vitro exposure to DEX or by in vivo exposure to thyroxine, but are increased by PHA and in some individuals by TNBS. When released from blockade, their thymic apoptotic rates rise as progress through metamorphosis is renewed. Larval thymic apoptosis is glucocorticocoid- and thyroxine insensitive, but is lectin and altered-self antigen activated, particularly during postclimax stages.

IN SITU LYMPHOCYTE APOPTOSIS IN LARVAL XENOPUS LAEVIS, THE SOUTH AFRICAN CLAWED TOAD

During Anuran metamorphosis larval structures regress, adult structures form anew and impaired T cell immune functions are noted, as are alterations in endogenous glucocorticoid titers. In situ histological data, after staining for DNA fragmentation, reveal patterns of lymphocyte suicide in the thymus and spleen of non-antigenically challenged, laboratory bred, developing larvae, that do not correlate with either impaired immune functions or plasma glucocorticoid titers. Apoptotic levels in the thymus are high in premetamorphic stages, low during prometamorphosis and high again, after metamorphic climax, reflecting a periodic removal of thymocytes. Lymphocytic apoptosis in the spleen is low during premetamorphosis, rises in prometamorphic stages, principally within the red pulp, reaching a peak at climax, before declining as metamorphosis is completed.

Phosphatidylserine expression on apoptotic lymphocytes of Xenopus laevis, the South African clawed toad, as a signal for macrophage recognition

Inflammation is avoided in apoptosis by early removal of dying cells by macrophages (MOs). In mammalian cells, an early aspect of apoptosis is the translocation of phosphatidylserine (PS) from the inner leaflet of the cell membrane to the surface. PS recognition can serve as a signal for triggering removal of dying cells. PS expression on splenocytes and thymocytes of Xenopus laevis was quantified using FITC-Annexin and flow cytometry following exposure in vitro to several known apoptogens for this species. All apoptogens used induced PS expression. Dose dependency and the kinetics of PS expression following exposure to the calcium ionophore, A23187, were also examined. Peritoneal exudate cells (PECs) were cultured with A23187-treated thymocytes to test MO capacity for recognition of PS. MO binding to apoptotic thymocytes was reduced following exposure of PECs to a water soluble analogue of PS, phospho-L-serine. The presence of a phagocytic PS-dependent recognition system in amphibia is supportive of the evolutionary conservation of this function in mammals that is crucial in limiting inflammation induced by dying cells.

Neuroendocrine regulation of immunity: the effects of noradrenaline in Xenopus laevis, the South African clawed toad.

A functional association between the peripheral nervous and the immune system in Xenopus laevis, the South African clawed toad, is demonstrated. This association involves the neurotransmitter noradrenaline (NA), produced and released by the sympathetic nerves of the spleen. Chemical sympathectomy prior to immunization reduces splenic NA, and decreases thymus-dependent (TD), but increases thymus-independent (TI), antibody responses. Immune challenge with representatives of the three antigen classes affects splenic NA levels differentially. Thus, the modulatory effect of NA on immunity will depend on the immunogen used. Carrier-priming of helper function in TD responses stimulates a transitory NA release in the spleen, while subsequent immunization activates a more prolonged release. The two types of challenge differ in the antigenic dose given. The effects of NA also depend on the time when it is applied. If used early in the in vivo TD response, antibody production is increased, but if given later, suppressor function is stimulated, thus decreasing antibody production. NA increases both amplifying and suppressing T cell functions in TD responses through stimulation of the alpha 2 adrenoceptor. Alpha 2 adrenoceptor stimulation decreases, and beta adrenoceptor stimulation increases, anti-TNP reactivity. Since an alpha 2 receptor agonist does not affect lectin-stimulated T cell mitogenesis, while a beta receptor agonist depresses it, NA appears to up-regulate T cell functions by affecting their maturation, rather than their clonal expansion.

The time course, localization and quantitation of T- and B-cell mitogen-driven apoptosis in vivo.

Apoptosis has very recently been visualized in situ in a mammalian thymus and spleen. We report here the first in situ visualization, localization and quantitation of the time course of mitogen-altered basal levels of apoptosis within the thymus and spleen of a vertebrate. Adult Xenopus leavis, South African clawed toads, were injected intraperitoneally with either the T-cell mitogen, Concanavalin (Con) A, or the B-cell mitogen, lipopolysaccharide (LPS). Controls, reflecting the basal level of apoptosis of both organs, were injected with isotonic phosphate-buffered saline for amphibians (APBS). ConA and LPS failed to enhance the nearly 2% apoptotic rate in the thymic cortex, which is made up largely of immature lymphocytes, but it did double the base level of 2% apoptosis in the mature lymphocytes of the medulla, particularly along the corticomedullary boundary. In the lymphoid splenic white pulp, the 2% basal level was exceeded slightly after ConA treatment, while the 6% basal lymphoid apoptotic rate in the red pulp was enhanced 7-fold in 12 h. LPS induced lymphocytic apoptosis in the partly erythropoietic red pulp of the spleen after 12 h but did not effect the white pulp. Extensive macrophage engulfment of apoptotic cells was apparent in both the thymus and the spleen.

Impaired T cell functions during amphibian metamorphosis: IL-2 receptor expression and endogenous ligand production

T cell functions are impaired during defined developmental stages of amphibian metamorphosis (Marx et al., 1987). Here we show, using a fluorescent anti-human IL-2 receptor antibody and flow cytometry, that during these stages, the splenocytes of Xenopus laevis, the South African clawed toad, have a progressively diminished capacity to express IL-2 receptors (IL-2R), after in vitro lectin stimulation. Preincubation with human rIL-2 specifically blocks binding of the anti-IL-2R antibody. Separation of an endogenous ligand bound to the IL-2R leads to a substantial increase in available epitope recognized by the anti-IL-2R antibody when pre- and postmetamorphic splenocytes are employed, but not when splenocytes of the prometamorphic stages are treated similarly. Thus, the cells from the prometamorphic stages are not producing significant quantities of the ligand. Finally, we demonstrate that human rIL-2 is not by itself mitogenic in the toad, but it can act as a co-stimulator of antigen-induced mitogenesis. Thus, an absence of an endogenous ligand (autologous IL-2?), coupled with a reduced capacity to express IL-2 receptors may be responsible for impaired T cell clonal expansion in metamorphosing Xenopus. Inhibition of T cell functions during this period is vital, since adult cells forming within the larval body bear surface proteins not found on larval cells (Flajnik et al., 1986).

Adrenergic Modulation of Apoptosis in Splenocytes of Xenopus laevis in vitro

Impaired and healthy cells undergo suicide using an intrinsic genetic program. Exposure to stress-related α2- or β-adrenergics for 4 or 20 h in vitro had no effect on apoptosis in splenocytes of adult Xenopus laevis, while a 4-hour coincubation of clonidine, an α2-agonist, with a calcium ionophore (A23187) or a phorbol diester (PMA), enhanced apoptosis induced by each apoptogen alone. Clonidine did not affect apoptosis stimulated with dexamethasone (DEX), however. Comparable in vitro exposures to isoproterenol, a β-agonist, reduced apoptotic levels stimulated by all three apoptogens alone. Following 20 h coexposure, clonidine no longer affected A23187-induced apoptosis, but reduced PMA-induced apoptosis, while isoproterenol enhanced apoptosis stimulated with both. Neither agonist modulated apoptosis induced by 20 h of exposure to DEX. Thus, adrenergic agonists modulated apoptosis in cells coexposed to A23187 and PMA, in a time-dependent and adrenoceptor class-dependent fashion. These stress-induced products can affect concurrent apoptosis reversibly over time in vitro, and thus possibly in vivo.

Splenic lymphocytes of adult Xenopus respond differentially to PMA in vitro by either dying or dividing: Significance for cancer resistance in this species

AbstractWild-type populations of amphibians, unlike mammalians, appear to be resistant to spontaneous and chemically induced neoplasms. Few true cancers have been reported for non-isogeneic members of Xenopus laevis, despite their widespread use in laboratories around the world. Injection of even the most powerful direct mammalian oncogens e.g. N-methyl N-nitrosourea, that depleted specific populations of T lymphocytes, did not induce cancer. Phorbol diesters, e.g. PMA, are mitogens and apoptogens in both amphibian, and mammalian immunocytes. In mammalian cells, regulation of the cell cycle and of apoptosis are often intimately linked, however, a disjunction in time between early apoptosis and later cell cycling, has been observed with PMA-treated Xenopus splenocytes. Thus, a particular difference between amphibians and mammals may be the requirement to enter the cell cycle before a progression to death by apoptosis. This hypothesis was tested here using dual staining flow cytometry. Xenopus laevis splenocytes were cultured for 8, 24 and 48 hours with phorbol 12-myristate 13-acetate (PMA), previously shown to be mitogenic and apoptotic with mature Xenopus lymphocytes. The cells were stained with FITC-conjugated Annexin V or with FITC-labeled deoxyuridine triphosphates (FITC-dUTP) to assay for the apoptotic markers phosphotidylserine or DNA strand breaks respectively. Phycoerythrin (PE)-conjugated anti-human proliferating cell nuclear antigen (PE-PCNA) was used as a cell cycle marker that is present during the entire cell cycle. Propidium iodide (PI) binds DNA and was used to assay for late stage apoptosis, as well as to assess DNA content.Significantly higher levels of apoptosis develop rapidly in PMA-exposed splenocytes and are maintained at 24 hours, declining by 48 hours. Cells expressing PCNA or incorporating PI in excess of the normal genomic level were found by 48 hours following PMA exposure. The absence of any significant rise in a small (<5%) dual staining cell population indicates that the apoptotic cell population remained distinct from cells already in the cell cycle from the onset of PMA exposure. Thus, Xenopus splenocytes respond differentially to PMA. Those that undergo apoptosis rapidly were quiescent, non-cycling small lymphocytes. Moreover, the cells that eventually begin division, following PMA exposure, were unaffected by the early apoptois and do not themselves die while in the cell cycle. The rapid apoptotic response of X. laevis cells to PMA may confer a natural cancer resistance in this species, as cells that fail to enter the cell cycle after exposure to cancer promoting reagents cannot express genetic destabilization that might have led to transformation.