Immunotoxic organotins as possible model compounds in studying apoptosis and thymocyte differentiation

TOXiCOlOGY ELSEVIER

Toxicology91 (1994) 189-202

Immunotoxic organotins as possible model compounds in studying apoptosis and thymocyte differentiation R.H.H. Pieters *a, M. Bol a, A.H. Penninks a'b aResearch Institute of Toxicology/Immunotoxicology Section, Utrecht University, P.O. Box 80.176, NL-3508 TD Utrecht, The Netherlands bTNO, Toxicology and Nutrition Institute, P.O. Box 360, NL-3700AJ Zeist, The Netherlands

(Received 13 September 1993; accepted 3 January 1994)

Abstract

In the mid-seventies it appeared that some organotin compounds selectively caused thymus atrophy. From that time onward efforts were made to reveal molecular and cellular mechanisms involved. In this review recent studies into organotin-sensitive stages and processes of thymocyte maturation are discussed. Together these studies resulted in the recognition of organotin compounds as possible model compounds in studying immature thymocyte differentiation and protein synthesis-independent apoptotic cell death of thymocytes. Keywords: Organotins; Immunotoxicology; Thymus; Apoptosis

1. Introduction

Organotin compounds were first synthesized by Farkland in 1849 (Van der Kerk, 1976). About a hundred years later, patents describing the use of dialkyltin derivatives as PVC stabilizers were granted to Ungve - in the early 1940s (Van der Kerk, 1976). Approximately 10 years later, tributyltin (TBT) compounds, in particular bis(tributyltin)oxide (TBTO), appeared being very useful as biocides in anti-fouling paints, agriculture and other uses (Van der Kerk, 1976; Snoeij et al., 1987). Over the years, applications of organotins have been extended with the use as industrial * Corresponding author. 0300-483X/94/$07.00 © 1994 Elsevier ScienceIreland Ltd. All rights reserved SSDI 0300-483X(94)02793-T

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catalysts (especially dialkyltins), in wood preservation (tributyltins and non-biocidal monobutyl- and monooctyltins) and as hospital and veterinary disinfectants (trialkyltins) (Van der Kerk, 1976; Snoeij et al., 1987; Penninks et al., 1990). From the 1950s, some organotins have been considered to be either neurotoxic (Magee et al., 1957; Snoeij et al., 1987; Penninks et al., 1990) hepatotoxic (Barnes and Magee, 1958; Snoeij et al., 1987; Penninks et al., 1990) or dermatotoxic (Snoeij et al., 1987). It was not until the early and mid-seventies that Seinen and Willems (1976) surprisingly found that di-n-octyltin dichloride (DOTC) was immunotoxic to rats. This effect was previously overlooked probably because of the fact that the knowledge of the immune system was rarely incorporated in protocol toxicology at that time. During the past two decades, many studies have focussed on the adverse effects of organotins on the immune system. Special attention has been paid to elucidation of the molecular mechanism of their selective thymotoxic activity. Because of the large amount of information available on the effects of organotins on the immune system they are regarded nowadays as immunotoxicological model compounds with a selective suppressive effect on T-cell development and function.

1.1. Thymus atrophy and impairment of T-cell-dependent immune functions Organotins, in particular dibutyltins (DBT) (Seinen et al., 1977), dioctyltins (DOT) (Seinen and Willems, 1976; Seinen et al., 1977) and tributyltins (TBT) (Snoeij et al., 1985; Krajnc et al., 1984) cause a reduction of thymus weight and cellularity in small rodents. In rats fed organotins for over 2 weeks a depletion of T-cell areas in spleen (PALS) and lymphnodes (paracortex) was also observed, although these effects were less profound when compared to those on the thymus (Seinen and Willems, 1976; Seinen et al., 1977; Snoeij et al., 1985; Krajnc et al., 1984). Thus, the thymus appears to be the most sensitive immune organ with respect to several diand trisubstituted organotins. Concomitantly with the depletion of peripheral T-cells, T-cell-dependent immune functions like allograft rejection (Seinen et al., 1977), delayed type hypersensitivity to tuberculin (Seinen et al., 1977) or ovalbumin (Vos et al., 1984), antibody formation against SRBC (Seinen et al., 1977) and GvH reactivity (Seinen et al., 1979) were impaired, while the T-cell-independent antibody formation towards LPS (Vos et al., 1984; Seinen et al., 1977) was not affected in organotin-treated animals. In TBTtreated rats a decreased NK cell activity was also observed (Vos et al., 1984). These general immunotoxic effects of organotins have been extensively reviewed recently (Penninks et al., 1990; Boyer, 1989; Nicklin and Robson, 1988). Therefore, this review is focussed on recent ideas regarding the mechanisms involved in the induction of thymus atrophy and on future research on organotins.

1.2. Characteristics of organotin-induced intrathymic changes and the possible target cell Morphometric measurements on sections of thymi from DBTC-treated and control rats that were stained with the cortical thymocyte-specific mAb HIS44 (Aspinall et al., 1991) showed a decrease of the cortical, but not medullary region of the thymus (Fig. 1). This finding supports previous similar observations on conventionally

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stained thymic sections (Seinen and Willems, 1976) and strongly indicates that the atrophy is restricted to the thymic cortex. Cell-size analyses combined with ex-vivo 3H-TdR-incorporation studies (Snoeij et al., 1988) on various days after single oral exposure indicated that DBTC-treatment initially results in a depletion of proliferating thymoblasts which already appeared maximal 24 to 48 h after treatment. The number of small, non-cycling thymocytes and thymus weight appeared to reach a minimum on days 4 to 5 after treatment (Fig. 2). Taken together, thymic weight reduction and depletion of small thymocytes are likely to be secondary to an arrest of thymocyte proliferation. Thymocyte maturation can be regarded as a sequence of maturation stages, or subsets, which can be distinguished phenotypically. Based on the lymphocyte surface markers CD4 and CD8, thymocytes are roughly subdivided into four main subsets being either C D 4 - C D 8 - , CD4÷CD8 ÷, C D 4 - C D 8 + or CD4+CD8 - (Nikolic-Zugic, 1991; Scollay et al., 1984, 1988). The most immature of these, the C D 4 - C D 8 - thymocytes, differentiate into CD4+CD8 + thymocytes which, in rat, has been shown to proceed via a transient immature, TcRa/~-low-positive C D 4 - C D 8 + stage (Scollay et al., 1988; Paterson and Williams, 1987). The CD4+CD8 ÷ cells, which constitute

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about 85% of all thymocytes (Scollay et al., 1984, 1988), either survive the intrathymic selection processes and become mature TcRa/3-high-positive CD4-CD8 + or CD4+CD8 - cells or die intrathymically (Scollay et al., 1984, 1988, Von Boehmer et al., 1989). About half of the CD4-CD8- thymocytes express the CD2 receptor (Williams et al., 1987) which can therefore be regarded as an early marker, while the CD5 marker is not expressed until the CD4+CD8 ÷ stage (Aspinall et al., 1991) and is thus a late differentiation marker (see Fig. 3 for an overall simplified scheme). Knowledge of the processes that regulate various steps of thymocyte differentiation has greatly increased over the past few years (Nikolic-Zugic, 1991). In order to further elucidate the cellular and molecular aspects of organotin-induced thymus atrophy, it was necessary to characterize the phenotype of the initially depleted thymoblasts. Detailed flow cytometric characterization of the sequential intrathymic changes after single oral DBTC treatment of rats showed that immature CD4CD8 + and CD4+CD8 ÷ thymoblasts expressing high levels of CD2 and no or low numbers of TcR# molecules on their membranes were initially depleted by 48 h (Fig. 2) (Pieters et al., 1992). A maximum reduction of the large population of small CD4+CD8 + thymocytes (Fig. 2), however, was not seen until days 4 to 5 (Pieters et al., 1992). Immunohistological stainings of thymic sections of similarly treated rats revealed a subsequent decrease of cortical cells positive for CD2, CD8, CD4, and CD5 (Pieters et al., 1989). This order of decrease was equal to the order of appearance of these markers during thymus ontogeny in the rat (Aspinall et al., 1991).

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Results together indicate that DBTC initially affects the formation of immature thymocyte subsets. The very immature C D 4 - C D 8 - subset, however, appeared numerically unaffected by DBTC (Pieters et al., 1992) supporting the idea that thymocyte depletion was not the result of a decreased immigration of pre-T-lymphocytes (Penninks et al., 1985). In vitro studies were performed to compare the effects of DBTC on the spontaneous and PHA-stimulated proliferation and on the differentiation of purified immature CD4-CD8 ÷ and C D 4 - C D 8 - thymocytes (Pieters et al., 1994). It appeared that the extent of inhibition of DNA-synthesis by DBTC was dependent on the proliferation rate in case of immature CD4-CD8 ÷, but not in case of purified C D 4 - C D 8 thymocytes, while the in vitro differentiation of both of these subsets into CD4 ÷ CD8 ÷ cells was anaffected by the chemical. The kinetics of changes in CD4/CD8subset distribution combined with changes in cell-size distribution in thymi of rats fed DBTC for 14 days (Pieters et al., 1994), revealed a continuous inhibition of proliferation but not of differentiation of immature CD4-CD8 ÷ thymocytes. Thus, in vivo findings appear to be in line with in vitro findings suggesting that DBTC selectively inhibits proliferation, but not differentiation of immature CD4-CD8 ÷ thymocytes. In the in vivo situation, DBTC treatment will result in a diminished

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replenishment of the more mature CD4+CD8 ÷ subset, while previously formed cells of this large CD4+CD8 + subset continue their differentiation undisturbed (Fig. 3). The time period of about 3 days between the maximum decrease of immature CD4-CD8 ÷ thymoblasts and of small CD4+CD8 + thymocytes and thymus weight after single DBTC treatment (Fig. 2) is in line with this idea, since the turnover time of CD4+CD8 ÷ thymocytes has been shown to be 3 days also (Scollay et al., 1988). In order to elucidate the molecular mechanism by which organotins induce thymus atrophy it remains to be established why the immature CD4-CD8 ÷ thymocyte proliferation is selectively sensitive to these agents. 1.3. Possible mechanisms involved in organotin-induced thymus atrophy

Proliferation as well as differentiation of thymocytes has been shown to depend on the receptor-dependent interaction with thymic epithelial cells, macrophages and interdigitating cells (van Ewijk, 1991). Interactions between cells and receptormediated activation have been shown to depend on sulfhydryl (SH)-containing molecules, which make these processes potentially vulnerable to the SH-reactive organotin compounds (Pfeifer and Irons, 1985). Dialkyltins, in particular, such as DBTC have been shown to display a strong affinity for dithiol groups (Fig. 4) (Davies and Smith, 1980; Penninks and Seinen, 1983) and may thus interfere with receptor-dependent communication between intrathymic cells. The CD2/LFA-3 receptor-ligand couple has been suggested to play a role in the adhesion of thymocytes with thymic epithelial cells (Vollger et al., 1987) thereby facilitating immature polyclonal thymocyte proliferation (Blue et al., 1987; Rubin et al., 1992; Fox et al., 1985). Interestingly, DBTC has been shown to inhibit the rosetring of human thymocytes with sheep red blood cells (Seinen et al., 1979), which

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depends on CD2/LFA-3 (Selvaraj et al., 1987). This, together with the findings that the thymoblasts that were initially depleted in DBTC-treated rats appeared to express high levels of CD2 (Pieters et al., 1992), gave rise to the hypothesis that DBTC inhibits immature thymocyte proliferation through interference with the CD2/LFA3-dependent interaction of thymocytes with thymic epithelial cells. Recently (Pieters et al., submitted), we have shown that DBTC is indeed capable of inhibiting the binding between freshly isolated thymocytes and thymic epithelial cells of the IT45R1 cell line (Itoh et al., 1981). However, it is not known as yet whether CD2 and LFA-3 (CD59, or the possible rat CD2-1igand CD48 (van der Merwe et al., 1993)), or other receptor/ligand couples play a role in this in vitro binding. The classical antigen-recognizing T-cell receptor-complex, TcRot#-CD3, is of pivotal importance in T-cell differentiation and selection (Blackman et al., 1990). This receptor-complex is expressed in high numbers predominantly on late CD4÷CD8 + and mature single positive thymocytes, while low numbers of TcRet/~ molecules were detected on early CD4+CD8 ÷ and also on immature CD4-CD8 ÷ thymocytes (Blue et al., 1987; Huniq, 1988). Importantly, cross-linking of these few molecules on immature CD4-CD8 ÷ thymocytes with an anti TcR/~ monoclonal antibody prevented their spontaneous overnight differentiation into CD4+CD8 + cells (H/inig, 1988; Takahama and Singer, 1992). From these studies, as well as from studies with TcR/~deficient transgenic mice showing an arrested differentiation of CD4-CD8- into CD4+CD8 ÷ thymocytes (Mombaerts et al., 1992), it was suggested that TcR/~ molecules play an important role in this early phase of thymocyte differentiation. For these reasons we tested the effect of DBTC on TcR/3-mediated signal transduction in freshly isolated thymocytes (Pieters, 1992). Surprisingly, DBTC dosedependently stimulated the initial Ca 2+ release elicited by cross-linking of TcR/3. This increased [Ca2+]i seems unrelated to apoptosis (see below), since in that case a sustained elevation of [Ca2+]i was to be expected (McConkey et al., 1989b). Interestingly, a similar effect on TcR/~-signalling was observed in case of cytochalasin B (Matsuyama et al., 1991), suggesting that it may result from an interference with cytoskeleton-dependent processes (Schreiner and Unanue, 1976; Braun and Unanue, 1983; Geppert and Lipsky, 1991). Preliminary unpublished experiments showing that DBTC delays the capping of TcR/~ but also of CD8 molecules on thymocytes may support this idea. How this effect relates to the observed anti-proliferative activity of DBTC is unknown as yet but interference with the cytoskeleton might also disturb the proper function of adhesion molecules and thereby the binding between thymocytes and TEC. Cytokines, which are produced by TEC, macrophages and thymocytes (Carding et al., 1991), and thymic hormones, which are produced by TEC (Zatz et al., 1982; Dardenne and Bach, 1988; Volsen et al., 1989), have been shown to be involved in various steps of thymocyte differentiation. Therefore, one might suggest that organotins interfere with the formation of these humoral factors. In thymocyte lysates from rats that were exposed to a single oral dose of DOTC, Volsen et al. (1989) observed a decrease of messenger RNA for IL2 which was maximal by 72 h after treatment. Concomitantly, they found a decrease of MHC class I-positive (OX18÷), probably medullary, thymocytes. They noted that immature thymocyte prolif-

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eration may well be influenced as a consequence of a reduced IL2 production by medullary thymocytes. However, it has been shown that immature thymocytes do not respond to IL2 (Boyer and Rothenberg, 1988; Ueno et al., 1989), while it is doubtful whether IL2 plays a major role in immature thymocyte differentiation at all (Takacs et al., 1988). Therefore, the observed decrease in intrathymic IL2 production may be of minor importance in respect of the mechanism of organotin-induced thymus atrophy. The production of other humoral factors has not been studied in relation to organotins. Immunohistologically, a shrinkage of cortical epithelial meshwork and an increase in cortical EDI ÷ and ED2 ÷ macrophage was observed, while electron microscopy showed an increased vacuolisation of the epithelial cells (Penninks et al., 1985). However, these changes were most profound by days 4 to 5 after single oral DBTC or DOTC treatment and therefore probably the result of the disappearance of interjacent thymocytes. Taken together, it seems unlikely that TEC, macrophages and consequently the production of humoral factors by these cells are directly involved in organotin-induced thymus atrophy. Clearly, despite much effort the molecular mechanism by which organotins, apparently very selectively cause thymus atrophy is still not clear. This may be partly due to the complexity of the process of thymocyte differentiation, but also to the difficulty to relate in vitro and in vivo effects. Various findings together, however, suggest that organotins may have an effect at the level of the cell membrane and/or cytoskeleton resulting in disturbances of inter- and intracellular communication processes, which are of crucial importance to thymocyte maturation.

1.4. ls apoptosis involved in organotin-induced thymus atrophy? In the young adult mice, 30% of all thymocytes are estimated to be newly produced every day while within the same period only 1% survives the selection process and leaves the thymus as immunocompetent T-cells (Scollay et al., 1984, 1988). Meanwhile 29% of all thymocytes are thought to die because they are potentially autoreactive or useless for the host defense system (Scollay et al., 1984, 1988; Von Boehmer et al., 1989). The process by which these cells die is known as apoptosis or active cell death (MacDonald and Lees, 1990). This physiological dying process is characterized by a typical endonuclease-controlled fragmentation of DNA followed by a gradual disintegration of cells and generally depends on protein and RNA synthesis (Green and Cotter, 1992). Apoptosis, which seems very important under a wide variety of physiological circumstances (Cotter, 1992) can be induced in thymocytes by corticosteroids (Cohen and Duke, 1984), anti-TcRot/3-CD3 antibodies (McConkey et al., 1989; Shi et al., 1991; Smith et al., 1989), but also by certain immunotoxic chemicals including TCDD (McConkey et al., 1988) and organotins (Aw et al., 1990; Raffray and Cohen, 1991). The similarity between corticosteroids (Screpanti et al., 1989), anti-TcRot/~-CD3 antibodies (Shi et al., 1991) and organotins with respect to the observed phenotypic thymic changes may suggest that organotin compounds cause thymus atrophy by increasing apoptotic cell death. It is important to mention in this regard that the organotin-induced thymus atrophy is not likely to be mediated by corticosteroids (Seinen and Willems, 1976; Snoeij et al., 1985; Krajnc et al., 1984).

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In vitro, TBT-compounds in particular, have been shown to cause apoptosis in thymocytes, measured as increased DNA fragmentation (Aw et al., 1990; Raffray and Cohen, 1991) and as a shift from CD4highCD8hightO CD41°wCD8I°wthymocytes (Pieters, 1992). DBTC appeared less effective in inducing apoptosis (Pieters, 1992). On an equimolar base, however, the thymic atrophy induced by DBTC is more pronounced when compared to TBTC (Snoeij et al., 1988). Moreover, intraveneous treatment with TBTC does not cause thymus atrophy, while DBTC is readily detectable in the blood within 3 h after single oral TBTC treatment (Snoeij et al., 1987). These findings gave rise to the assumption that TBTC-induced thymus atrophy in rats is caused by its proposed metabolite DBTC (Snoeij et al., 1988). Recently, however, Raffray and Cohen (1993) have detected an increased DNA fragmentation in thymocyte suspensions isolated from TBT-treated rats from which they concluded that TBT, unlike DBT, induces thymocyte depletion by apoptosis. This fragmentation, measured as DNA fragments present in the supernatant of ultracentrifuged thymocytes, was found to increase from 10 /~g supernatant DNA per 108 cells in controls to 15 #g at 18 h after dosing and to about 45 #g at 48 h (Raffray and Cohen, 1993). However, since the number of thymocytes in TBT-treated rats was about three times lower than in controls, the DNA fragmentation can be calculated to be only about 150% of controls on a total thymic organ base. In addition, from findings showing that TBTC and DBTC (Snoeij et al., 1988) have similar anti-proliferative effects, a decrease of newly produced non-apoptotic CD4+CD8 + thymocytes, like that noted in DBTC-treated rats, has to be expected in TBTC-exposed rats as well. This will result in a relative increase of previously formed, already negativelyselected and thus apoptotic cells. Taken together, it may be doubted whether apoptosis is of major importance to the mechanism of TBT-induced thymus atrophy. Yet, it cannot be excluded that, in addition to inhibition of proliferation, organotins cause a more severe DNA fragmentation among immature thymoblasts, an effect that cannot be detected in total thymocyte suspensions due to the large background apoptosis. Clearly, additional studies are required to establish whether organotins induce apoptosis in specific thymic subpopulations. Nevertheless, much research needs to be done to reveal the exact mechanisms of apoptosis involved under various circumstances. An interesting finding was that protein- and ATP-synthesis, which are both processes profoundly inhibited by TBT (Raffray et al., 1993; Snoeij et al., 1986), appeared not necessary for TBT-induced apoptosis to occur (Raffray et al., 1993). Although the mechanism of the proteinsynthesis-independent apoptosis is not fully understood as yet, constitutively present endonucleases are likely to be involved (Cotter, 1992; Wyllie, 1980). Regardless of the outcome of the studies regarding the role of apoptosis in organotin-induced thymus atrophy, compounds such as TBT may be very useful model compounds to study the biochemistry of the protein-synthesis-independent apoptosis in particular. 1.5. Organotin-compounds and thymocyte development

As discussed above, organotins cause a characteristic change in thymocyte subset distribution resulting from a depletion of immature thymoblasts. Organotin compounds thereby stand out from other thymolytic compounds, such as the halogen-

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ated aromatic hydrocarbon 2,3,7,8-TCDD, which acts probably at the TEC or prothymocyte level (Schuurman et al., 1991); cyclosporin A, which disturbs the differentiation step from CD4+CD8 + into mature single positive thymocytes (Jenkins et al., 1988); and the food additive Caramel Colour III, which causes a decrease in cortical thymocytes, a reduced expression of ED2 on cortical macrophages and an increase in the number of medullary thymocytes (Houben et al., 1991). Research on the mechanisms involvec~ in the thymic effects of these chemicals may yield important fundamental information on the process of thymocyte differentiation. With regard to organotins, in particular the finding that the initially-depleted immature CD4-CD8 ÷ thymoblasts as well as the thymoblasts responsible for repopulation of the organotin-depleted thymus express high levels of CD2 (Pieters et al., 1993) may be of relevance not only to the understanding of the mechanism of organotininduced thymus atrophy but also of the role of CD2 in immature thymocyte differentiation (Rubin et al., 1992). Interestingly, during recovery from organotin-induced thymus atrophy a moderate increase in the number of TcRaB+CD4-CD8 thymoblasts was detected (Pieters et al., 1993) while others have found that activation of immature thymoblasts through CD2 also results .in an increased formation of these normally rare cells (Tiefenthaler et al., 1992). The role of these cells is not completely known as yet, but their expansion in human and mouse SLE suggests a role in certain forms of autoimmunity (Reimann, 1991). Therefore, it may be useful to examine whether cells with this particular phenotype appear in the periphery during or after recovery from organotin-induced thymus atrophy. I f so, the organotinmodel may be useful to examine the role of the thymus in the formation of these rare T-cells. In summary, organotin compounds, which are regarded as model compounds in immunotoxicology, may be very useful in studying fundamental aspects of the process of apoptotic cell death and of immature thymocyte development.

2. References Aspinall, R., Kampinga, J. and Van den Bogaerde, J. (1991) T-cell development in the fetus and the invariant series hypothesis. Immunol. Today 12, 7. Aw, T.Y., Nicotera, P., Manzo, L. and Orrenius, S. (1990) Tributyltin stimulates apoptosis in rat thymocytes. Arch. Biochem. Biophys. 283, 46. Barnes, J.M. and Magee, P.M. 0958) The biliary and hepatic lesion produced experimentally by dibutyltin salts. J. Pathol. Bacteriol. 75, 267. Blackman, M., Kappler, J. and Marrack, P. (1990) The role of the T-cell receptor in positiveand negative selection of developing T-cells, Science 248, 1335. Blue, M.-L., Daley, J.F., Levine, H., Craig, K.A. and Schlossman, S.F. (1987a) Activation of immature cortical thymocytesthrough the T11 sheep erythrocyte binding protein. J. Immunol. 138, 3108. Blue, M.-L., Daley,J.F., Levine,H., Craig, K.A. and Schlossman, S.F. (1987b) Identificationof T4+T8+ cells with high T3 expression in human thymus: a possible late intermediate in thymocytedifferentiation. J. Immunol. 139, 1065. Boyer, P.D. and Rothenberg, E.V. (1988) II-2receptor inducibility is blocked in cortical-typethymocytes. J. Immunol. 140, 44. Boyer, I.J. 0989) Toxicologyof dibutyltin, tributyltin and other organotin compounds to human and experimental animals. Toxicol. 55, 253.

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