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

Wild-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 apoptosis 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.

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.

Apoptosis and the cell cycle in Xenopus: PMA and MPMA exposure of splenocytes.

Spontaneous and induced cancers are rare in non-isogeneic or inbred amphibians. Neoplastic cells become immortalized through loss of a normal capacity to die by apoptosis. Mature lymphocytes of mammals require activation and entry into the cell cycle in order to become susceptible to apoptosis. Whether Xenopus lymphocytes differ from mammalian lymphocytes in this regard is examined. In vitro exposure of PMA, or its analogue, MPMA, to adult splenocytes of Xenopus laevis was used to affect apoptosis. Flow cytometric analysis of FITC-Annexin V/propidium iodide (PI) fluorescence (apoptosis) and BrdU uptake (DNA synthesis) were assayed concurrently in the same lymphocyte population over time. Significant increases in apoptotic levels were induced throughout a 72 hour period in PMA-treated cells only. Lymphocytes were also separated by size for analysis. Several subpopulations of lymphocytes were identified, the most interesting of which was small and apoptotic within 4 hours, after PMA exposure. PMA-induced DNA synthesis did not become elevated until after 24 hours. "Direct" apoptosis, i.e. without cell cycle entry, was found only in these small, mature lymphocytes. Since small lymphocytes make up the vast majority of those being analyzed, "direct" apoptosis may be a determining mechanism in the resistance to neoplasia observed in Amphibia. Cells that die more readily are less likely to transform into neoplastic cells.

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.

Apoptosis and the cell cycle in Xenopus laevis: PHA and PMA exposure of splenocytes.

T cell receptor (TCR) ligation and protein kinase C (PKC) activation stimulate proliferation and modulate apoptosis in both mammalian and amphibian lymphocytes. The potential relationship between apoptosis and the cell cycle in mature Xenopus laevis splenic lymphocytes is addressed by monitoring apoptosis and DNA synthesis over time, using incorporation of propidium iodide (PI) and flow cytometry. Aliquots of the same populations of cells are followed after exposure in vitro to phytohemagglutinin (PHA) or phorbol 12-myristate 13-acetate (PMA). Significant increases in apoptosis preceed those in DNA synthesis by 12 to 16 h following exposure to both reagents. Since apoptosis preceeds DNA synthesis, these dying cells clearly do not need to enter the S phase of the cell cycle before becoming apoptotic, in contrast to mammalian T cells. Another striking difference is that the reagent with weaker mitogenic properties in this species, PHA, is significantly a more potent apoptogen, than the strong mitogen, PMA. The two phenomena then appear to be inversely related in Xenopus cells. Data on DNA synthesis suggest independence of the two phenomena, as DNA synthesis is stimulated in direct proportion to the strength of each reagent as a mitogen. Mature mammalian T-cells undergo apoptosis only when previously activated. The Xenopus lymphocytes examined were not deliberately activated by exposure to antigen or lectin. PMA, a cancer promoter in mammals, usually 'rescues' mammalian cells from apoptosis, but stimulates apoptotic increases in Xenopus cells. Thus, mature Xenopus lymphocytes may be more readily stimulated to die by cancer inducing agents than mammalian lymphocytes. This could make them less susceptible to transformation into immortalized cancer cells. This characteristic may considerably contribute to the observed resistance to spontaneous and chemically-induced neoplasia in wild type, non-isogeneic or non-inbred Xenopus.

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.

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.

Interleukin-2-induced mortality during the metamorphosis of Xenopus laevis.

In anuran metamorphosis, histoincompatible adult cells arise within an immunocompetent larval body. However, the larvae are unresponsive to these altered-self cells. The basis for this tolerance is an issue of considerable interest. While a loss of tolerance in mammalian pregnancy may initiate localized abortion, since the entire metamorphic amphibian is involved, there is the potential for total body self-destruction. Metamorphosing Xenopus laevis, the South African clawed toad, produce an internal corticosterone environment that induces T-cell anergy. This impairment may save the animal from immune self-destruction. Here we examine the capacity of recombinant gene produced human interleukin 2 (IL-2) to substitute for, or restore the level of autologous IL-2, as a further test of whether the altered-self tolerance found during metamorphosis may rely on corticosteroid-induced anergy. We find that the capacity of rIL-2 to break this tolerance and stimulate mortality is low, unless it is accompanied by antigenic co-stimulation. A study of sections of experimental and control animals revealed lymphocyte and mast cell increases within the kidney, particularly in the region of the coelomoduct, perhaps reflecting autoimmune reactivity responsible for the mortality.

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.

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.

A comparison of the effects of human rIL-2 and autologous TCGF on Xenopus laevis splenocytes in vitro.

Prior studies have shown that intraperitoneal (ip) injection of 25 IU of human rIL-2 can effectively modulate in vivo immune reactivity to thymus-dependent and thymus-independent type 2 immunogens in Xenopus laevis, the South African clawed toad, but is less successful at affecting toad cells in vitro. Here we compare the capacities of human rIL-2 and autologous TCGF to modulate Xenopus splenocytes in vitro and find that autologous TCGF (1) is more effective at stimulating mitogenesis, (2) can serve as a ligand for inducible receptors that will also bind rIL-2 and an F1*-mouse anti-human p55 antibody, and (3) will regulate the expression these receptors.

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.

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.

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).

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.

Thymus-replacing activity from the metamorphic spleen of Xenopus laevis.

Anuran metamorphosis offers an interesting vertebrate immunological paradigm, for adult cells that arise within the immunocompetent larval body are MHC Class I disparate from those of the larva. The animals, in order to avoid immune self-destruction during this transition period, are made unresponsive to these modified-self cells by an impairment of T-cell functions. However, it remains to be discovered how an animal with compromised T-cell functions can protect itself from those environmental pathogens protection from which is thymus dependent. During metamorphosis, larval and adult immunocytes capable of reacting to each other coexist within the animal. Their interaction might stimulate the secretion of a cytokine capable of circumventing T-cell functions by acting directly on B cells. Here, we report that such an activity is released in vitro by metamorphic and not by adult splenocyte suspensions. This activity will amplify in vitro anti-hapten responses by immunized, but not carrier-primed, adult splenocytes. The activity is unaffected by dexamethasone and, since it will amplify anti-hapten responses in T-cell-depleted immunized adult splenocyte suspensions, the antibody-producing (B) cell population may be affected directly. Two radiolabeled protein peaks of 65 kD and 40 kD were obtained by SDS-PAGE analysis from secreting, metamorphosing, but not from adult, splenocytes.

Postnatal transmission of AIDS-associated retrovirus from mother to infant.

The third child of a previously healthy woman was delivered by caesarean section. Because of intraoperative blood loss, a blood transfusion was given after the delivery. The baby was breast-fed for 6 weeks. One unit of blood came from a male in whom the acquired immunodeficiency syndrome (AIDS) developed 13 months later. On recall, the mother proved to have lymphadenopathy, serum antibody to the AIDS virus, and a reduced T4/T8 ratio. The infant, who failed two thrive and had atopic eczema from 3 months, has likewise proved to have antibody to the AIDS virus. Since his mother was transfused after his birth, he is presumed to have been infected via breast milk or by way of some other form of close contact with his mother.

Pigmentary macular degeneration with multifocal necrotizing encephalopathy.

A previously healthy 10-year-old girl suffered sudden, binocular visual deterioration. During the next few years her neurologic and visual condition progressively worsened and she developed hypertension, seizures, ataxia, and lactic acidemia, leading to death at the age of 16 years. Bilateral optic disk pallor was followed by the loss of the foveal reflex and pigmentary maculopathy, manifested as disorganization of the retinal layers, loss of ganglion cells, degeneration of the photoreceptors and nuclei, and irregular infiltration of the retina by pigment epithelial cells. The optic nerves and tracts showed central axonal loss. Bilateral, multifocal symmetric areas of cerebral atrophy and necrosis of the neuropil and neurons in the cerebral cortex, basal ganglia, and thalamus were observed; neurons persisted in the dorsal medulla, despite neuropil degeneration.

Slit-lamp examination of the bedridden patient.

Slit-lamp examination of bedridden patients is facilitated by using a stretcher with the patient lying prone and holding the head erect.