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.

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PubMedDate not supplied

The development of peripheral TNP-tolerance and suppressor function in Xenopus laevis, the South African clawed toad.

In adult Xenopus laevis, inducer- and effector-suppressor functions are located in the spleen. These peripheral suppressor functions must be established at this location near the end of metamorphosis, since both functions are in the thymus in premetamorphic and in developmentally-blocked metamorphosing larvae. This study examined whether TNP-conjugated self-antigens resulting from exposure to trinitrobenzene sulfonic acid (TNBS), will stimulate TNP-tolerance in premetamorphic, metamorphic, and in developmentally-blocked metamorphosing larvae. Premetamorphic and developmentally-blocked larvae produce little TNP-tolerance or peripheral suppressor function. However, when TNBS exposure includes the late stages of the metamorphic period, both TNP-tolerance and splenic anti-hapten suppressor function are demonstrable. Removal of suppressor function with cyclophosphamide prevents expression of tolerance, thus, they are functionally related. Suppressor function and tolerance both differentiate during the late metamorphic stages when new adult antigens are being expressed and incorporated into a library of self.

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PubMedDate not supplied

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 (PEC's) were cultured with A23187-treated thymocytes to test MO capacity for recognition of PS. MO binding to apoptotic thymocytes was reduced following exposure of PEC's 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.

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PubMedDate not supplied

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.

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PubMedDate not supplied

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.

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PubMedDate not supplied

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 alpha2- or beta-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 alpha2-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 beta-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.

Adrenergic alpha-2 Receptor Agonists↗