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Chemicals trophic for the thymus: Risk for immunodeficiency and autoimmunity
0192-0561/92 $5.00 + ,00 Pergamon Press plc. International Society for Immunopharmacology.
Int. J. hnrnunopharmac., Vok 14, No. 3, pp. 369-375, 1992.
Printed in Greal Britain.
C H E M I C A L S T R O P H I C FOR THE T H Y M U S : RISK FOR I M M U N O D E F I C I E N C Y A N D A U T O I M M U N I T Y HENK-JAN
S C H U U R M A N , *~* H E N K V A N L O V E R E N , *
JAN ROZ1NG ~
a n d JOSEPH G. VOS*
*Laboratory for Pathology, National Institute of Public Health and Environmental Protection, Bilthoven; *Division of Histochemistry and Electron Microscopy, Departments of Internal Medicine and Pathology, University Hospital, Utrecht; and ~Department of Immunology, TNO-Institute for Aging and Vascular Research, Leiden, The Netherlands
Abstract - - The thymus is considered as the privileged site of T-lymphocyte generation. The organ is extremely vulnerable to the toxic action of chemicals. The classical example is the "acute stress-induced involution" mediated by glucocorticoid steroid hormones from the adrenal cortex. Nowadays a number of substances have been identified that act in a differential way on the thymus. Examples presented are some organotin compounds acting on immature lymphoblasts in the outer cortex, glucocorticosteroids acting on small thymocytes in the cortex, 2,3,7,8-tetrachlorodibenzo-p-dioxin acting on epithelial cells in the cortex, and cyclosporin acting on dendritic cells and epithelium in the medulla. The mechanisms of toxicity include receptor binding (Ah, aryl hydrocarbon receptor; dioxin); the Ca -'~-dependent activation of an endogenous endonuclease resulting in DNA fragmentation ("programmed cell death" or apoptosis; dioxin and glucocorticosteroids); and interference with cell proliferation (some organotin compounds). The consequences of toxicity can be a decrease in thymic output of newly-generated T-lymphocytes (i.e. generation of a new T-cell repertoire), or induction of autoimmune symptoms by the creation of unwanted repertoire. This latter phenomenon may be applicable to cyclosporin that under specified conditions can induce so-called syngeneic graft-vs-host disease. This survey presents a brief description of the function of the thymus and the various thymic cell populations involved. Thereafter the susceptibility to toxic insults and the mechanisms of toxicity are reviewed. Finally, the consequences of toxic action for the host defence system, and hence the health status, are considered.
The extreme sensitivity o f the t h y m u s to i m m u n o t o x i c chemical substances was recognized five decades ago, w h e n Selye (1936) described the effects o f stress o n the i m m u n e system. In these studies, the basis o f thymic i n v o l u t i o n was directly associated with the synthesis o f glucocorticoid steroid h o r m o n e s by the a d r e n a l cortex. This sensitivity o f the t h y m u s to chemical substances (such as stress h o r m o n e s ) was established long before its f u n c t i o n as a p r i m a r y l y m p h o i d o r g a n in the immune system was discovered. This early o b s e r v a t i o n o f t h y m i c sensitivity to stress h o r m o n e s is best u n d e r s c o r e d by the m a g n i t u d e of the reaction. Firstly, the s h r i n k i n g of the o r g a n is macroscopically visible. Secondly, this shrinkage or i n v o l u t i o n becomes visible already within days after the insult.
Thirdly, the h i s t o p a t h o l o g y shows characteristic changes (Van Baarlen et al., 1988), e.g. the a p p e a r a n c e o f starry-sky m a c r o p h a g e s followed by l y m p h o c y t e depletion o f the cortex a n d shrinking o f the thymic lobules with a n increase in thickness of the interlobular septae. In the discipline o f i m m u n o t o x i c o l o g y the t h y m u s has a central position ( S c h u u r m a n et al., 1991b). However, w h e n using the t h y m u s as a read-out system for general i m m u n o t o x i c i t y studies, two aspects should be kept in m i n d . Firstly, in line with the a b o v e example, stress should be avoided in the experimental testing a n d evaluation, since it has a m a i n effect o n thymus. Secondly, the t h y m u s involutes with age, which reduces the effect of toxic action in animals at older age. The " a c u t e
*Author to whom correspondence should be addressed at: Laboratory for Pathology, National Institute of Public Health and Environmental Protection, P.O. Box 1, 3720 BA Bilthoven, The Netherlands. 369
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involution" differs from the "physiological" ageassociated involution in many aspects, not only in speed but also in histologic features. In this paper we will approach the vulnerability of the thymus to toxic action from a number of viewpoints. We will first present a brief description of the function of the thymus and the various cell populations involved. This is followed by the susceptibility of these cell populations to toxic insults and the mechanisms of toxicity. Finally, the consequences of toxic action for the status of the host defence system, and hence the health status, are considered.
F U N C T I O N OF T H E T H Y M U S
In the lymphoid system, the thymus is considered as the privileged site of T-lymphocyte generation (Blackman et al., 1990; Sprent et al., 1990; Von Boehmer & Kisielow, 1990). A main feature of this T-cell development is the expression of the antigenrecognizing unit, the so-called T-cell receptor (TCR). Prothymocytes, entering the organ from the bone marrow, generally are negative in TCR-expression: the process of rearrangement of genes encoding the receptor starts within the thymic microenvironment. The sole expression of TCR is not an indication of maturity, because after surface TCR-expression the cells are subjected to selection. Positive selection allows "passage" of cells recognizing antigen in the context of the polymorphic determinants of the major histocompatibility complex (MHC). Negative selection stops cells recognizing auto-antigens. The process of gene rearrangement is not unique for lymphocytes within the thymus, as it is concluded to occur outside the organ in conditions of e.g, congenital thymic aplasia (e.g. athymic nude animals). However, the process of selection is unique for the thymus and results in the generation of the "functional" repertoire from the totally-available repertoire. In other words: only those cells that have the proper potential to recognize and react to antigens (positive selection) but not to potential auto-antigens (negative selection), emerge from the total pool comprising all possible TCR-entities and are allowed to leave for the periphery. Also, the new T-cell repertoire is generated in this way. The epithet " u n i q u e " has been added after the description of selection processes, i.e. the process from the totalavailable repertoire into functional repertoire including clonal selection on the basis of MHCrestriction-specificity and clonal deletion on the basis
of autoreactive potential. In addition the thymus has an endocrine function, by the synthesis and secretion of biologically active thymic hormones, such as thymosin and thymulin (Dardenne & Bach, 1988). CELL POPULATIONS IN THE THYMUS Each lobule of the thymus is composed of a subcapsular cortex, cortex and medulla (Kendall, 1988). These compartments differ in the population of both stationary (stroma) and passenger cells (leukocytes) (Table 1). The stroma is composed of reticular epithelial cells. This frame-work component is unique, as it does not occur in other lymphoid organs. Epithelial cells in the cortex and medulla differ in morphology and marker expression, which might be related to the different embryonic origin of these cells. The passenger leukocyte populations definitely belong to different lineages. Macrophages occur in the cortex and medulla. In addition, the medulla harbours a special cell type belonging to the monocyte lineage, the so-called interdigitating reticulum cell (IDC). The main function of macrophages is phagocytosis, whereas that of IDC is the presentation of antigens. Also, the lymphocyte populations have different characteristics in different compartments. A rough subdivision includes the subcapsular area and outer cortex, where lymphocytes are in the most immature stage (the so-called "prothymocyte" stage); the cortex, where cells are in an immature stage of maturation; and the medulla lodging lymphocytes in the most mature stage of development. This differentiation is reflected by differences in cytology and immunologic phenotype such as TCR-expression. There is no TCR-expression in subcapsular lymphoblasts, and the TCR is low in density on cells with the cortex phenotype and is high in density on medullary-type lymphocytes. It is generally assumed that the selection process following TCR-gene rearrangement involves the close interaction of the developing T-cells with the unique microenvironment including the epithelium and IDC (thereby shaping the unique function of the thymus in the immune system). The functional aspects of the thymus are further discussed below, in relation to the consequences of toxic damage to the individual's immune capacity. T A R G E T CELLS FOR TOXICITY
The unique composition of cell types, which is not found in other lymphoid organs, underlies the distinct susceptibility of the thymus to toxic damage
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Table 1. Cell populations in the thymus, and their sensitivity to toxic chemicals Cells
Location
Target function
Lymphocytes Immature blasts Small thymocytes CD4+CD8' Intermediate lymphocytes CD4+CD8 or CD4-CD8 +
Outer cortex Cortex Medulla
Some organotin compounds Glucocorticosteroids
Epithelial cells Cortex (endoderm) Medulla (ectoderm)
Cortex Medulla
2,3,7,8-tetrachlorodibenzo-p-dioxin Cyclosporin A ?
Dendritic cells
Medulla
Cyclosporin A, FK-506
Macrophages
Cortex, medulla
(Schuurman et al., 1991b). Essentially, all cell populations can be the target of the action of toxic chemicals, but show differences in their susceptibility to different compounds (Table 1). This will be illustrated by some examples: (1) Substances like certain immunotoxic organotin compounds have action first on lymphocytes of the thymus (Krajnc et al., 1984; Penninks et al., 1991; Van Loveren et al., 1991). It has been claimed that the large lymphoblasts in the outer cortex are the main target of toxicity (Pieters et al., 1989; Snoeij et al., 1988). (2) Glucocorticosteroid hormones also affect lymphocytes. Glucocorticosteroid-sensitive lymphocytes encompass all lymphocytes of the cortex. This phenomenon is related to the histopathology of cortical changes in acute stress involution described above. (3) Substances like halogenated aromatic hydrocarbons affect the epithelial component, especially reticular epithelial cells in the cortex (Van Loveren et al., 1991; Schuurman et al., 1991a). This has been documented in in vitro studies using epithelial cell cultures (Cook et al., 1987; Greenlee et al., 1985), but also in studies on the histopathology of the thymus after exposure to e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin (Schuurman et al., 1991a; De Waal et al., manuscript submitted). In the latter approach on normal rats, we found indications for a shift in epithelial cells from pale large-sized cells (with signs of activity) towards intermediate cells (showing signs of degeneration). In addition, changes in immunophenotype of the epithelial cells in the shrunken cortex did occur. An increase was noted of epithelial cells with a "double-positive"
phenotype, expressing marker molecules that in the normal uninvoluted thymus only occur on cortex epithelium or on subcapsular/medullary epithelium. Such cells occur only sporadically in the normal thymus. These "double-positive" cells may represent a form of degeneration or dedifferentiation. In addition to their effects on epithelial cells, there are indications that halogenated aromatic hydrocarbons directly affect lymphocytes of the thymus. This has been documented during in vitro cultures, generally at high concentrations of the compound (McConkey et al., 1988). Also an effect on bone marrow T-cell precursors has been described, which represents an indirect effect on thymic alterations (Fine et al., 1990). (4) Finally, interdigitating cells and epithelial cells in the medulla can be the target for drugs like cyclosporin A (CsA) (Beschorner et al., 1987; Schuurman et al., 1990) and FK-506 (PughHumphreys et al., 1990), which are applied as immunosuppressive agents in prevention of e.g. organ transplant rejection reactions. The interest in the thymic effects of these drugs arose, when under experimental conditions in rodents unexpected autoimmune-like reactions could be evoked by these compounds, indicating a possible defective repertoire generation in the thymus (Glazier et al., 1983). We and others (Beschorner et al., 1987; Schuurman et al., 1990) noted the disappearance of IDC in thymus of rats after CsA treatment. The above-mentioned examples illustrate that nowadays a spectrum of toxic compounds able to interfere with distinct cell populations of the thymus is available. This bears relevance not only for toxicologically oriented studies (e.g. the study of the
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mechanism of action of different compounds on the thymus), but also for immunologically oriented studies (e.g. manipulating different components of the thymus to evaluate their function).
MECHANISMS OF TOXICITY Receptors. The susceptibility of thymic cells to toxicological and pharmacological substances is due to the expression of relevant receptors inside the cell or on the cell surface. Glucocorticosteroid (stress) hormones, for instance, affect cortical lymphocytes, because these cells express hormone receptors. Sex hormones and hormone analogues are worth mentioning in this respect. Their suppressive effect has been demonstrated, e.g. by the increase in thymic size after gonadectomy both in males and females (Greenstein et al., 1986). Analogues of luteinizing hormone-releasing hormone induce thymic hypertrophy, or thymic regeneration of an ageinvoluted thymus (Greenstein et al., 1987). On the contrary, the increased production of sex hormones during pregnancy has been related to the involution of the thymus during this period (Clarke & Kendall, 1989). These actions are assumed to be mediated by hormone receptors, which for a number of glucocorticoid hormones have been demonstrated on thymic cells. The susceptibility to aromatic hydrocarbons like 2,3,7,8-tetrachlorodibenzo-pdioxin is related to the presence of the aryl hydrocarbon (Ah) receptor, which occurs especially in thymic epithelial cells (Cook et al., 1987; Poland & Knutson, 1982). Intrinsic metabolic maRe-up. The intrathymic selection process includes interaction with the microenvironment resulting in the " g o " signal in positive selection, and the "stop" signal and cell death in negative selection. Related to this phenomenon thymocytes will die in situ when not appropriately stimulated, or die when stimulated during negative selection. A peculiar response after stimulation is the Ca2+-dependent activation of an endogenous endonuclease, resulting in DNA fragmentation and cell lysis. This process is called "programmed-cell death" or apoptosis (Duvall & Wyllie, 1986). It has received increasing attention during the last decade as a mechanism of cell death. Thus, dependent on the metabolic make-up of the cell, the same stimulus and the same target cell can result on the one hand in lysis by apoptosis, and on the other hand in stimulation, differentiation and
proliferation. This has been well illustrated for ligands interfering with the TCR (Smith et al., 1989; Murphy et al., 1990): in immature lymphocytes, like thymus cortex cells, binding results in apoptosis, and in mature ("immunocompetent") cells this results in stimulation and cell division. Thus, apoptosis represents a physiologic process occurring amongst others during negative selection in T-celt maturation. Those clones that are not negatively selected pass through and develop the metabolic make-up that enables them to make a proliferative response when stimulated by the same type of ligand (antigen + MHC) in the periphery. Apoptosis is also the mechanism of toxicity for a number of immunotoxic substances. It has been demonstrated in vitro for thymic lymphocytes under the influence of glucocorticosteroid stress hormones (McConkey et al., 1989), sex hormones, and 2,3,7,8tetrachlorodibenzo-p-dioxin (McConkey et al., 1988). These compounds differ in histopathologic features of thymic involution that have to be related to their action on different cell types (glucocorticosteroids mainly lymphocytes, dioxins mainly epithelium) and to the density of receptor expression. The absence of an overt toxicity by lymphocytes in other compartments than the thymus cortex has to be explained by the fact that these cells react differently after ligand binding to the receptor. The fragile character of immature lymphocytes in the thymus, underlying their vulnerability to toxic insult, it is also related to their make-up of purine nucleotide metabolic enzymes. Due to this enzyme make-up, cells accumulate toxic intermediates during the process of DNA synthesis, resulting in cell death of proliferating cells (Cohen et al., 1980; Schuurman et al., 1983). It has been hypothesized that appropriate rescue is provided by the microenvironment (Ma et al., 1983). Substances with anti-proliferative action may interfere with proliferating lymphocytes in the outer cortex. Examples are organotin compounds and ionizing irradiation (Anderson et al., 1986). For such toxic insults thymic involution is not associated with an increase of apoptotic figures. Rather, it is explained by blocking proliferation at the earliest stage of lymphocyte maturation yielding the depletion of progeny in the cortex. The cell biologic basis of toxic action to other cell types of the thymus remains largely unknown. Epithelium in the cortex reacts to 2,3,7,8tetrachlorodibenzo-p-dioxin by a shift from an active cell (with a pale nucleus in ultrastructure) into a more inactive, presumably degenerating, cell (with an intermediate appearance in the electron microscope),
Chemicals Trophic for the Thymus and the appearance of an immunologic phenotype presumably associated with "dedifferentiation". The process following Ah-receptor binding yielding this response is unresolved. Medullary epithelium, and more particular IDC in the medulla, disappear under the influence of CsA and FK-506. This, apparently, is a special feature of the stromal cells of the thymus: for instance IDC in lymphoid organs like the spleen do not disappear under influence of CsA. CONSEQUENCES OF THYMUS TOXICITY The consequences of thymus toxicity should be considered with regard to the unique functions of the organ described above. Thus, the first effect is immunosuppression, related to the interference with the generation of a new repertoire. This effect is most obvious in conditions when the repertoire is produced, i.e. during embryogenesis and in early life (Luster et al., 1980; Vos & Moore, 1974). For experimental and clinical situations this concerns e.g. T-cell regeneration after bone marrow transplantation. It is well established that the thymus in the normal adult individual shows a very low output of newly-generated T-cells (Scollay et al., 1980, 1986), associated with age-associated involution (Schuurman et al., 1991c). Thus, for immunotoxicology it can be questioned whether toxic insults to the thymus after reaching adulthood have any sense for the immune status, in broader terms the health status, of the individual. In other words, risks for immunodeficiency by thymus toxicity are only apparent during the first life period, and in case that there is a simultaneous action with elimination of the peripheral pool (e.g. in the situation after bone marrow transplantation). The second consequence of thymus toxicity for the health status regards the interference with only part of the intrathymic processes, for instance with selection. The interference with positive selection (adaptation to MHC-restriction-specificity) essentially can be not harmful, in the sense that it loads the individual with a nonsense repertoire. Such a situation may exist for T-like cells in athymic animals (McDonald et al., 1987; Vaessen et al., 1986). We have as yet no examples for such a condition in immunotoxicology. However, compounds such as 2,3,7,8-tetrachlorodibenzo-p-dioxin affect cortical epithelial cells which have a function in positive selection. It might therefore be relevant to analyse peripheral T-cells after exposure to this compound for the presence of a nonsense repertoire.
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On the other hand, auto-immunity can be the result of interference with selection. The interference with negative selection can result in the export of cell populations that have not been depleted of potentially auto-reactive clones. The best example for such an activity comes from CsA treatment. Short-term CsA treatment after bone marrow transplantation results in auto-immune symptoms resembling graft-vs-host disease, even in case of syngeneic bone marrow transplantation. This phenomenon, called syngeneic graft-vs-host disease (Glazier et al., 1983), has been related to the CsAinduced disappearance of IDC from the thymus medulla, resulting in the absence of negative selection (Shi et al., 1989). Also in the case of prenatal and neonatal exposure to CsA, negative selection vanishes and potentially auto-reactive cells appear in the periphery. But, overt auto-immune disease symptoms do not occur in this situation, which may be ascribed to induction of tolerance by clonal anergy in the periphery (Sprent et al., 1990). So, CsA-induced auto-immune disease based on its thymus toxicity is only apparent in the case of a large export of newly-generated non-negatively-selected T-cells. The final effect of thymus toxicity on health status comes from its endocrine activity. A decreased level of circulating thymic hormone has been reported for people with presumed thymic damage after exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (Stehr-Green et al., 1989). The function of circulating thymic hormones in the endocrine circuit is not precisely known. Awaiting data on this aspect, a decreased thymic hormone level may be useful as a peripheral marker for thymus function rather than being an indicator for the effects of thymus alterations on the health status. In conclusion, the particular vulnerability of the thymus to toxic compounds renders the organ to be a sensitive indicator of early damage to the immune system. The consequences of thymus toxicity for immunodeficiency and auto-immunity are probably most significant at those moments in life when the organ displays a high activity level, e.g. in the natural situation of embryonic and early postnatal life period. In normal adulthood these effects, accordingly, are presumably less. However, the thymus output of a newly-generated T-cell repertoire even at old age does not become zero. This has advantages in the case of a need for newly-generated cells (after immunosuppression), but also has the dangerous potential of generating auto-reactive cells. So, even in later life with an involuted thymus thymic toxicity might yield negative results.
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H. -J. SCHUURMANet al. REFERENCES
ANDERSON, R. E., STANDEFER,J. C. • TOKUDA,S. (1986). The structural and functional assessment of cytotoxic injury of the immune system with particular reference to the effects of ionizing radiation and cyclophosphamide. Br. Z Cancer, 53 (Suppl. 7), 140- 160. BESCHORNER, W. E., NAMNOUN, J. D., HESS, A. D., SHINN, G. A. t~ SANTOS, G. W. (1987). Cyclosporin A and the thymus. Immunopathology. Am. J. Path., 126, 487-496. BLACKMAN, M., KAPPLER, J. & MARRACK,P. (1990). The role of the T cell receptor in positive and negative selection of developing T cells. Science, 248, 1335- 1341. CLARKE, A. G. & KENDALL, M. D. (1989). Histological changes in the thymus during mouse pregnancy. Thymus, 14, 65 - 78. COrtEN, A., LEE, J. W. W., DOSCH, H.-M. & GELFAND, E. W. (1980). The expression of deoxyguanosine toxicity in T lymphocytes at different stages of maturation. J. Immun., 125, 1578- 1582. COOK, J. C., DOLD, K. M. & GREENLEE, W. F. (1987). An in vitro model for studying the toxicity of 2,3,7,8tetrachlorodibenzo-p-dioxin to human thymus. Toxic. appl. Pharmac., 89, 256-268. DARDENNE, M. & BACH, J. F. (1988). Functional biology of thymic hormones. Thymus Update, 1, 101 - 116. DUVALL, E. & WYLLIE, A. H. (1986). Death and the cell. Immun. Today, 4, 115-119. FINE, J. S., SILVERSTONE, A. E. & GASIEWlCZ, T. A. (1990). Impairment of prothymocyte activity by 2,3,7,8tetrachlorodibenzo-p-dioxin. J. Immun., 144, 1169- 1176. GLAZIER, A., TUTSCHKA,P. J., FARMER,E. R. & SANTOS,G. W. (1983). Graft-versus-host disease in cyclosporin A-treated rats after syngeneic and autologous bone marrow reconstitution. J. exp. Med., 158, 1 - 8. GREENLEE, W. F., DOLD, K. M., IRONS, R. D. & OSBORNE, R. (1985). Evidence for direct action of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) on thymic epithelium. Toxic. appl. Pharmac., 79, 112-120. GREENSTEIN, B. D., FITZPATRICK, F. T. A., ADCOCK, 1. M. & KENDALL,M. D. (1986). Reappearance of the thymus in old rats after orchidectomy: inhibition of regeneration by testosterone. J. Endocrinol., liD, 417- 422. GREENSTE1N, B. D., FITZPATRICK,F. T. A., KENDALL,M. D. & WHEELER, M. J. (1987). Regeneration of the thymus in old male rats treated with a stable analogue of LHRH. J. Endocrinol., 112, 345- 350. KENDALL,M. D. (1988). Anatomical and physiological factors influencing the thymic microenvironment. Thymus Update, 1, 27 - 65. KRAJNC, E. I., WESTER, P. W., LOEBER, J. G., VAN LEEUWEN, F. X. R., Vos, J. G., VAESSEN,H. A. M. G. & VAN DER HEYDEN, C. A. (1984). Toxicity of bis(tri-n-butyltin)oxide in the rat. 1. Short-term effects on general parameters and on the endocrine and lymphoid system. Toxic. appl. Pharmac., 75, 363- 384. LUSTER, M. I., BOORMAN, G. A., DEAN, J. H., HARRIS, M. W., LUEBKE, R. W., PADARATHSINGH,M. el al. (1980). Examination of bone marrow, immunologic parameters and host susceptibility following pre- and postnatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Int. J. Immunopharmac., 2, 301- 310. MA, D. D. F., SYLWESTROWICZ,T., JANOSSY, G. & HOFFBRAND,A. V. (1983). The role of purine metabolic enzymes and terminal deoxynucleotidyl transferase in intrathymic T-cell differentiation, lmmun. Today, 4, 6 5 - 68. MCCONKEY, D. J., HARTZELL, P., DUDDY, S. K., H~KANSSON,H. & ORRENIUS, S. (1988). 2,3,7,8-Tetrachlorodibenzo-pdioxin kills immature thymocytes by CaZ'-mediated endonuclease activation. Science, 242, 256-259. MCCONKEY, D. J., NICOTERA, P., HARTZELL, P., BELLOMO, G., WYLLIE, A. H. & ORRENIUS, S. (1989). Glucocorticosteroids activate a suicide process in thymocytes through an elevation of cytosolic Ca 2 concentrations. Arch. Biochem. Biophys., 269, 365- 370. McDONALD, H. R., LEES, R. K., BRON, C., SORDAT, B. & MIESCHER, G. H. (1987). T cell antigen receptor expression in athymic (nu/nu) mice. Evidence for an oligoclonal/3 chain repertoire. J. exp. Med., 166, 195-209. MURPHY, K. M., HEIMBERGER, A. B. & LOH, D. Y. (1990). Induction by antigen by intrathymic apoptosis of CD4 ~ CD8' TCR m thymocytes in vivo. Science, 250, 1720- 1722. PENNINKS, A. H., DIETERS, R. H. H., SNOEIJ, N. J. & SEINEN, W. (1991). Organotin-induced thymus atrophy. Thymus Update, 4, 5 7 - 79. PIETERS, R. H. H., KAMPINCIA, J., BoL-SCHOENMAKERS,M., LAM, B. W., PENNINKS, A. H. & SEINEN, W. (1989). Organotin-induced thymus atrophy concerns the OX-44 + immature thymocytes. Thymus, 14, 79-88. POLAND, A. & KNUTSON, J. C. (1982). 2,3,7,8-Tetrachlorodibenzo-p-dioxin and related halogenated hydrocarbons: examination of the mechanism of toxicity. A. Rev. Pharmac. Toxic., 22, 517-554. PUGH-HUMPHREVS, R. G. P., Ross, C. S. K. & THOMSON, A. W. (1990). The influence of FK-506 on the thymus. An immunophenotypic and structural analysis. Immunology, 70, 398- 404. SCHUURMAN, H.-J., DE WAAL, E. J., VAN LOVEREN, H. & VOS, J. G. (1991a). The toxicity of dioxin to the thymus. Thymus Update, 4, 3 5 - 55. VAN LOVEREN, H., SCHUURMAN,H.-J., KAMPINGA,J. &; Vos, J. G. Reversibility of thymic atrophy induced by 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) and bis(tri-n-butyltin)oxide (TBTO). Int. J. hnmunopharmac., 13, 369-377.
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SCHUURMAN,H.-J, KRAJNC-FRANKEN,M. A. M., KUPER, C. F., VAN LOVEREN,H. & VOS, J. G. (1991b). Immune System. In Handbook o f Toxicologic Pathology (eds I-Iasheck-Hock, W. M. and Rosseaux, C. G.), pp. 421 -487. Academic Press, New York. SCHUURMAN,H.-J., NAGELKERKEN,L., DE WEGER, R. A. & ROZING, J. (1991c). Age-associated involution: significance of a physiologic process. Thymus, 18, 1 - 6 . SCHUURMAN,H. J., VAN LAARHOVEN,J. P. R. M., BROEKHU1ZEN,R., SPIERENBURG,G.Th., BREKELMANS,P., FIGDOR, C. G. & KATER, L. (1983). Lymphocyte maturation in the human thymus. Relevance of purine nucleotide metabolism for intrathymic T cell function. Scand. J. Immun., 18, 539-549. SCHUURMAN,H.-J., VAN LOVEREN,H., ROZlNG, J., VAN DIJK, A., LOEBER, J. G. & VOS, J. G. (1990). Cyclosporin and the rat thymus. An lmmunohistochemical study. Thymus, 16, 235- 254. SCOLLAV, R., BUTCHER, E. C. & WEISSMAN, I. L. (1980). Thymus cell migration. Quantitative aspects of cellular traffic from the thymus to the periphery in mice. Eur. J. lmmun., 10, 210-218. SCOLLAY,R., SMITH,J. • STAUFFER,V. (1986). Dynamics of early T cells: prothymocyte migration and proliferation in the adult mouse thymus, lmmun. Rev., 91, 129- 157. SELVE, H. (1936). Thymus and adrenals in the response of the organism to injuries and intoxications. Br. J. exp. Path., 17, 234. Sin, Y., SAHAI, B. M. & GREEN, D. R. (1989). Cyclosporin A inhibits activation-induced cell death in T-cell hybridomas and thymocytes. Nature, 339, 625- 626. SMITH, C. A., WILLIAMS, G. T., KINGSTON, R., JENKINSON, E. J. & OWEN, J. J. T. (1989). Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures. Nature, 337, 181 - 184. SNOE1J, N. J., PENNINKS,A. H. & SEINEN, W. (1988). Dibutyltin and tributyltin compounds induce thymus atrophy in rats due to a selective action on thymic lymphoblasts. Int. J. lmmunopharmac., 10, 891-899. SPRENT, J., GAO, E. K. & WEBB, S. R. (1990). T cell reactivity to MHC molecules: immunity versus tolerance. Science, 248, 1357- 1363. STEHR-GREEN, P. A., NAYLOR, P. H. & HOFFMAN, R. E. (1989). Diminished thymosino.~ levels in persons exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Toxic. Envir. Hlth, 28, 285-295. VAESSON, L. M. B., BROEKHUIZEN,R., ROZING, J., Vos, J. G. & SCHUURMAN,H.-J. (1986). T-cell development during ageing in congenitally athymic (nude) rats. Scand. J. lmmun., 24, 223- 235. VAN BAARLEN, J., SCHUURMAN,H.-J. & HUBER, J. (1988). Acute thymus involution in infancy and childhood: a reliable marker for duration of acute illness. Human Path., 19, 1155- 1160. VAN LOVEREN, H., SCHUURMAN,H.-J., KAMPINGA,J. & Vos, J. G. (1991). Reversibility of thymic atrophy induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and bis(tri-n-butyltin)oxide (TBTO). Int. J. Immunopharmac., 13, 369 - 377. YON BOEHMER, H. & KISIELOW, P. (1990). Self-nonself discrimination by T cells. Science, 248, 1369- 1372. Vos, J. G. & MOORE, J. A. (1974). Suppression of cellular immunity in rats and mice by maternal treatment with 2,3,7,8tetrachlorodibenzo-p-dioxin. Int. Arch. Allergy appl. Immun., 47, 777- 794.
Int. J. hnrnunopharmac., Vok 14, No. 3, pp. 369-375, 1992.
Printed in Greal Britain.
C H E M I C A L S T R O P H I C FOR THE T H Y M U S : RISK FOR I M M U N O D E F I C I E N C Y A N D A U T O I M M U N I T Y HENK-JAN
S C H U U R M A N , *~* H E N K V A N L O V E R E N , *
JAN ROZ1NG ~
a n d JOSEPH G. VOS*
*Laboratory for Pathology, National Institute of Public Health and Environmental Protection, Bilthoven; *Division of Histochemistry and Electron Microscopy, Departments of Internal Medicine and Pathology, University Hospital, Utrecht; and ~Department of Immunology, TNO-Institute for Aging and Vascular Research, Leiden, The Netherlands
Abstract - - The thymus is considered as the privileged site of T-lymphocyte generation. The organ is extremely vulnerable to the toxic action of chemicals. The classical example is the "acute stress-induced involution" mediated by glucocorticoid steroid hormones from the adrenal cortex. Nowadays a number of substances have been identified that act in a differential way on the thymus. Examples presented are some organotin compounds acting on immature lymphoblasts in the outer cortex, glucocorticosteroids acting on small thymocytes in the cortex, 2,3,7,8-tetrachlorodibenzo-p-dioxin acting on epithelial cells in the cortex, and cyclosporin acting on dendritic cells and epithelium in the medulla. The mechanisms of toxicity include receptor binding (Ah, aryl hydrocarbon receptor; dioxin); the Ca -'~-dependent activation of an endogenous endonuclease resulting in DNA fragmentation ("programmed cell death" or apoptosis; dioxin and glucocorticosteroids); and interference with cell proliferation (some organotin compounds). The consequences of toxicity can be a decrease in thymic output of newly-generated T-lymphocytes (i.e. generation of a new T-cell repertoire), or induction of autoimmune symptoms by the creation of unwanted repertoire. This latter phenomenon may be applicable to cyclosporin that under specified conditions can induce so-called syngeneic graft-vs-host disease. This survey presents a brief description of the function of the thymus and the various thymic cell populations involved. Thereafter the susceptibility to toxic insults and the mechanisms of toxicity are reviewed. Finally, the consequences of toxic action for the host defence system, and hence the health status, are considered.
The extreme sensitivity o f the t h y m u s to i m m u n o t o x i c chemical substances was recognized five decades ago, w h e n Selye (1936) described the effects o f stress o n the i m m u n e system. In these studies, the basis o f thymic i n v o l u t i o n was directly associated with the synthesis o f glucocorticoid steroid h o r m o n e s by the a d r e n a l cortex. This sensitivity o f the t h y m u s to chemical substances (such as stress h o r m o n e s ) was established long before its f u n c t i o n as a p r i m a r y l y m p h o i d o r g a n in the immune system was discovered. This early o b s e r v a t i o n o f t h y m i c sensitivity to stress h o r m o n e s is best u n d e r s c o r e d by the m a g n i t u d e of the reaction. Firstly, the s h r i n k i n g of the o r g a n is macroscopically visible. Secondly, this shrinkage or i n v o l u t i o n becomes visible already within days after the insult.
Thirdly, the h i s t o p a t h o l o g y shows characteristic changes (Van Baarlen et al., 1988), e.g. the a p p e a r a n c e o f starry-sky m a c r o p h a g e s followed by l y m p h o c y t e depletion o f the cortex a n d shrinking o f the thymic lobules with a n increase in thickness of the interlobular septae. In the discipline o f i m m u n o t o x i c o l o g y the t h y m u s has a central position ( S c h u u r m a n et al., 1991b). However, w h e n using the t h y m u s as a read-out system for general i m m u n o t o x i c i t y studies, two aspects should be kept in m i n d . Firstly, in line with the a b o v e example, stress should be avoided in the experimental testing a n d evaluation, since it has a m a i n effect o n thymus. Secondly, the t h y m u s involutes with age, which reduces the effect of toxic action in animals at older age. The " a c u t e
*Author to whom correspondence should be addressed at: Laboratory for Pathology, National Institute of Public Health and Environmental Protection, P.O. Box 1, 3720 BA Bilthoven, The Netherlands. 369
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involution" differs from the "physiological" ageassociated involution in many aspects, not only in speed but also in histologic features. In this paper we will approach the vulnerability of the thymus to toxic action from a number of viewpoints. We will first present a brief description of the function of the thymus and the various cell populations involved. This is followed by the susceptibility of these cell populations to toxic insults and the mechanisms of toxicity. Finally, the consequences of toxic action for the status of the host defence system, and hence the health status, are considered.
F U N C T I O N OF T H E T H Y M U S
In the lymphoid system, the thymus is considered as the privileged site of T-lymphocyte generation (Blackman et al., 1990; Sprent et al., 1990; Von Boehmer & Kisielow, 1990). A main feature of this T-cell development is the expression of the antigenrecognizing unit, the so-called T-cell receptor (TCR). Prothymocytes, entering the organ from the bone marrow, generally are negative in TCR-expression: the process of rearrangement of genes encoding the receptor starts within the thymic microenvironment. The sole expression of TCR is not an indication of maturity, because after surface TCR-expression the cells are subjected to selection. Positive selection allows "passage" of cells recognizing antigen in the context of the polymorphic determinants of the major histocompatibility complex (MHC). Negative selection stops cells recognizing auto-antigens. The process of gene rearrangement is not unique for lymphocytes within the thymus, as it is concluded to occur outside the organ in conditions of e.g, congenital thymic aplasia (e.g. athymic nude animals). However, the process of selection is unique for the thymus and results in the generation of the "functional" repertoire from the totally-available repertoire. In other words: only those cells that have the proper potential to recognize and react to antigens (positive selection) but not to potential auto-antigens (negative selection), emerge from the total pool comprising all possible TCR-entities and are allowed to leave for the periphery. Also, the new T-cell repertoire is generated in this way. The epithet " u n i q u e " has been added after the description of selection processes, i.e. the process from the totalavailable repertoire into functional repertoire including clonal selection on the basis of MHCrestriction-specificity and clonal deletion on the basis
of autoreactive potential. In addition the thymus has an endocrine function, by the synthesis and secretion of biologically active thymic hormones, such as thymosin and thymulin (Dardenne & Bach, 1988). CELL POPULATIONS IN THE THYMUS Each lobule of the thymus is composed of a subcapsular cortex, cortex and medulla (Kendall, 1988). These compartments differ in the population of both stationary (stroma) and passenger cells (leukocytes) (Table 1). The stroma is composed of reticular epithelial cells. This frame-work component is unique, as it does not occur in other lymphoid organs. Epithelial cells in the cortex and medulla differ in morphology and marker expression, which might be related to the different embryonic origin of these cells. The passenger leukocyte populations definitely belong to different lineages. Macrophages occur in the cortex and medulla. In addition, the medulla harbours a special cell type belonging to the monocyte lineage, the so-called interdigitating reticulum cell (IDC). The main function of macrophages is phagocytosis, whereas that of IDC is the presentation of antigens. Also, the lymphocyte populations have different characteristics in different compartments. A rough subdivision includes the subcapsular area and outer cortex, where lymphocytes are in the most immature stage (the so-called "prothymocyte" stage); the cortex, where cells are in an immature stage of maturation; and the medulla lodging lymphocytes in the most mature stage of development. This differentiation is reflected by differences in cytology and immunologic phenotype such as TCR-expression. There is no TCR-expression in subcapsular lymphoblasts, and the TCR is low in density on cells with the cortex phenotype and is high in density on medullary-type lymphocytes. It is generally assumed that the selection process following TCR-gene rearrangement involves the close interaction of the developing T-cells with the unique microenvironment including the epithelium and IDC (thereby shaping the unique function of the thymus in the immune system). The functional aspects of the thymus are further discussed below, in relation to the consequences of toxic damage to the individual's immune capacity. T A R G E T CELLS FOR TOXICITY
The unique composition of cell types, which is not found in other lymphoid organs, underlies the distinct susceptibility of the thymus to toxic damage
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Table 1. Cell populations in the thymus, and their sensitivity to toxic chemicals Cells
Location
Target function
Lymphocytes Immature blasts Small thymocytes CD4+CD8' Intermediate lymphocytes CD4+CD8 or CD4-CD8 +
Outer cortex Cortex Medulla
Some organotin compounds Glucocorticosteroids
Epithelial cells Cortex (endoderm) Medulla (ectoderm)
Cortex Medulla
2,3,7,8-tetrachlorodibenzo-p-dioxin Cyclosporin A ?
Dendritic cells
Medulla
Cyclosporin A, FK-506
Macrophages
Cortex, medulla
(Schuurman et al., 1991b). Essentially, all cell populations can be the target of the action of toxic chemicals, but show differences in their susceptibility to different compounds (Table 1). This will be illustrated by some examples: (1) Substances like certain immunotoxic organotin compounds have action first on lymphocytes of the thymus (Krajnc et al., 1984; Penninks et al., 1991; Van Loveren et al., 1991). It has been claimed that the large lymphoblasts in the outer cortex are the main target of toxicity (Pieters et al., 1989; Snoeij et al., 1988). (2) Glucocorticosteroid hormones also affect lymphocytes. Glucocorticosteroid-sensitive lymphocytes encompass all lymphocytes of the cortex. This phenomenon is related to the histopathology of cortical changes in acute stress involution described above. (3) Substances like halogenated aromatic hydrocarbons affect the epithelial component, especially reticular epithelial cells in the cortex (Van Loveren et al., 1991; Schuurman et al., 1991a). This has been documented in in vitro studies using epithelial cell cultures (Cook et al., 1987; Greenlee et al., 1985), but also in studies on the histopathology of the thymus after exposure to e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin (Schuurman et al., 1991a; De Waal et al., manuscript submitted). In the latter approach on normal rats, we found indications for a shift in epithelial cells from pale large-sized cells (with signs of activity) towards intermediate cells (showing signs of degeneration). In addition, changes in immunophenotype of the epithelial cells in the shrunken cortex did occur. An increase was noted of epithelial cells with a "double-positive"
phenotype, expressing marker molecules that in the normal uninvoluted thymus only occur on cortex epithelium or on subcapsular/medullary epithelium. Such cells occur only sporadically in the normal thymus. These "double-positive" cells may represent a form of degeneration or dedifferentiation. In addition to their effects on epithelial cells, there are indications that halogenated aromatic hydrocarbons directly affect lymphocytes of the thymus. This has been documented during in vitro cultures, generally at high concentrations of the compound (McConkey et al., 1988). Also an effect on bone marrow T-cell precursors has been described, which represents an indirect effect on thymic alterations (Fine et al., 1990). (4) Finally, interdigitating cells and epithelial cells in the medulla can be the target for drugs like cyclosporin A (CsA) (Beschorner et al., 1987; Schuurman et al., 1990) and FK-506 (PughHumphreys et al., 1990), which are applied as immunosuppressive agents in prevention of e.g. organ transplant rejection reactions. The interest in the thymic effects of these drugs arose, when under experimental conditions in rodents unexpected autoimmune-like reactions could be evoked by these compounds, indicating a possible defective repertoire generation in the thymus (Glazier et al., 1983). We and others (Beschorner et al., 1987; Schuurman et al., 1990) noted the disappearance of IDC in thymus of rats after CsA treatment. The above-mentioned examples illustrate that nowadays a spectrum of toxic compounds able to interfere with distinct cell populations of the thymus is available. This bears relevance not only for toxicologically oriented studies (e.g. the study of the
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mechanism of action of different compounds on the thymus), but also for immunologically oriented studies (e.g. manipulating different components of the thymus to evaluate their function).
MECHANISMS OF TOXICITY Receptors. The susceptibility of thymic cells to toxicological and pharmacological substances is due to the expression of relevant receptors inside the cell or on the cell surface. Glucocorticosteroid (stress) hormones, for instance, affect cortical lymphocytes, because these cells express hormone receptors. Sex hormones and hormone analogues are worth mentioning in this respect. Their suppressive effect has been demonstrated, e.g. by the increase in thymic size after gonadectomy both in males and females (Greenstein et al., 1986). Analogues of luteinizing hormone-releasing hormone induce thymic hypertrophy, or thymic regeneration of an ageinvoluted thymus (Greenstein et al., 1987). On the contrary, the increased production of sex hormones during pregnancy has been related to the involution of the thymus during this period (Clarke & Kendall, 1989). These actions are assumed to be mediated by hormone receptors, which for a number of glucocorticoid hormones have been demonstrated on thymic cells. The susceptibility to aromatic hydrocarbons like 2,3,7,8-tetrachlorodibenzo-pdioxin is related to the presence of the aryl hydrocarbon (Ah) receptor, which occurs especially in thymic epithelial cells (Cook et al., 1987; Poland & Knutson, 1982). Intrinsic metabolic maRe-up. The intrathymic selection process includes interaction with the microenvironment resulting in the " g o " signal in positive selection, and the "stop" signal and cell death in negative selection. Related to this phenomenon thymocytes will die in situ when not appropriately stimulated, or die when stimulated during negative selection. A peculiar response after stimulation is the Ca2+-dependent activation of an endogenous endonuclease, resulting in DNA fragmentation and cell lysis. This process is called "programmed-cell death" or apoptosis (Duvall & Wyllie, 1986). It has received increasing attention during the last decade as a mechanism of cell death. Thus, dependent on the metabolic make-up of the cell, the same stimulus and the same target cell can result on the one hand in lysis by apoptosis, and on the other hand in stimulation, differentiation and
proliferation. This has been well illustrated for ligands interfering with the TCR (Smith et al., 1989; Murphy et al., 1990): in immature lymphocytes, like thymus cortex cells, binding results in apoptosis, and in mature ("immunocompetent") cells this results in stimulation and cell division. Thus, apoptosis represents a physiologic process occurring amongst others during negative selection in T-celt maturation. Those clones that are not negatively selected pass through and develop the metabolic make-up that enables them to make a proliferative response when stimulated by the same type of ligand (antigen + MHC) in the periphery. Apoptosis is also the mechanism of toxicity for a number of immunotoxic substances. It has been demonstrated in vitro for thymic lymphocytes under the influence of glucocorticosteroid stress hormones (McConkey et al., 1989), sex hormones, and 2,3,7,8tetrachlorodibenzo-p-dioxin (McConkey et al., 1988). These compounds differ in histopathologic features of thymic involution that have to be related to their action on different cell types (glucocorticosteroids mainly lymphocytes, dioxins mainly epithelium) and to the density of receptor expression. The absence of an overt toxicity by lymphocytes in other compartments than the thymus cortex has to be explained by the fact that these cells react differently after ligand binding to the receptor. The fragile character of immature lymphocytes in the thymus, underlying their vulnerability to toxic insult, it is also related to their make-up of purine nucleotide metabolic enzymes. Due to this enzyme make-up, cells accumulate toxic intermediates during the process of DNA synthesis, resulting in cell death of proliferating cells (Cohen et al., 1980; Schuurman et al., 1983). It has been hypothesized that appropriate rescue is provided by the microenvironment (Ma et al., 1983). Substances with anti-proliferative action may interfere with proliferating lymphocytes in the outer cortex. Examples are organotin compounds and ionizing irradiation (Anderson et al., 1986). For such toxic insults thymic involution is not associated with an increase of apoptotic figures. Rather, it is explained by blocking proliferation at the earliest stage of lymphocyte maturation yielding the depletion of progeny in the cortex. The cell biologic basis of toxic action to other cell types of the thymus remains largely unknown. Epithelium in the cortex reacts to 2,3,7,8tetrachlorodibenzo-p-dioxin by a shift from an active cell (with a pale nucleus in ultrastructure) into a more inactive, presumably degenerating, cell (with an intermediate appearance in the electron microscope),
Chemicals Trophic for the Thymus and the appearance of an immunologic phenotype presumably associated with "dedifferentiation". The process following Ah-receptor binding yielding this response is unresolved. Medullary epithelium, and more particular IDC in the medulla, disappear under the influence of CsA and FK-506. This, apparently, is a special feature of the stromal cells of the thymus: for instance IDC in lymphoid organs like the spleen do not disappear under influence of CsA. CONSEQUENCES OF THYMUS TOXICITY The consequences of thymus toxicity should be considered with regard to the unique functions of the organ described above. Thus, the first effect is immunosuppression, related to the interference with the generation of a new repertoire. This effect is most obvious in conditions when the repertoire is produced, i.e. during embryogenesis and in early life (Luster et al., 1980; Vos & Moore, 1974). For experimental and clinical situations this concerns e.g. T-cell regeneration after bone marrow transplantation. It is well established that the thymus in the normal adult individual shows a very low output of newly-generated T-cells (Scollay et al., 1980, 1986), associated with age-associated involution (Schuurman et al., 1991c). Thus, for immunotoxicology it can be questioned whether toxic insults to the thymus after reaching adulthood have any sense for the immune status, in broader terms the health status, of the individual. In other words, risks for immunodeficiency by thymus toxicity are only apparent during the first life period, and in case that there is a simultaneous action with elimination of the peripheral pool (e.g. in the situation after bone marrow transplantation). The second consequence of thymus toxicity for the health status regards the interference with only part of the intrathymic processes, for instance with selection. The interference with positive selection (adaptation to MHC-restriction-specificity) essentially can be not harmful, in the sense that it loads the individual with a nonsense repertoire. Such a situation may exist for T-like cells in athymic animals (McDonald et al., 1987; Vaessen et al., 1986). We have as yet no examples for such a condition in immunotoxicology. However, compounds such as 2,3,7,8-tetrachlorodibenzo-p-dioxin affect cortical epithelial cells which have a function in positive selection. It might therefore be relevant to analyse peripheral T-cells after exposure to this compound for the presence of a nonsense repertoire.
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On the other hand, auto-immunity can be the result of interference with selection. The interference with negative selection can result in the export of cell populations that have not been depleted of potentially auto-reactive clones. The best example for such an activity comes from CsA treatment. Short-term CsA treatment after bone marrow transplantation results in auto-immune symptoms resembling graft-vs-host disease, even in case of syngeneic bone marrow transplantation. This phenomenon, called syngeneic graft-vs-host disease (Glazier et al., 1983), has been related to the CsAinduced disappearance of IDC from the thymus medulla, resulting in the absence of negative selection (Shi et al., 1989). Also in the case of prenatal and neonatal exposure to CsA, negative selection vanishes and potentially auto-reactive cells appear in the periphery. But, overt auto-immune disease symptoms do not occur in this situation, which may be ascribed to induction of tolerance by clonal anergy in the periphery (Sprent et al., 1990). So, CsA-induced auto-immune disease based on its thymus toxicity is only apparent in the case of a large export of newly-generated non-negatively-selected T-cells. The final effect of thymus toxicity on health status comes from its endocrine activity. A decreased level of circulating thymic hormone has been reported for people with presumed thymic damage after exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (Stehr-Green et al., 1989). The function of circulating thymic hormones in the endocrine circuit is not precisely known. Awaiting data on this aspect, a decreased thymic hormone level may be useful as a peripheral marker for thymus function rather than being an indicator for the effects of thymus alterations on the health status. In conclusion, the particular vulnerability of the thymus to toxic compounds renders the organ to be a sensitive indicator of early damage to the immune system. The consequences of thymus toxicity for immunodeficiency and auto-immunity are probably most significant at those moments in life when the organ displays a high activity level, e.g. in the natural situation of embryonic and early postnatal life period. In normal adulthood these effects, accordingly, are presumably less. However, the thymus output of a newly-generated T-cell repertoire even at old age does not become zero. This has advantages in the case of a need for newly-generated cells (after immunosuppression), but also has the dangerous potential of generating auto-reactive cells. So, even in later life with an involuted thymus thymic toxicity might yield negative results.
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H. -J. SCHUURMANet al. REFERENCES
ANDERSON, R. E., STANDEFER,J. C. • TOKUDA,S. (1986). The structural and functional assessment of cytotoxic injury of the immune system with particular reference to the effects of ionizing radiation and cyclophosphamide. Br. Z Cancer, 53 (Suppl. 7), 140- 160. BESCHORNER, W. E., NAMNOUN, J. D., HESS, A. D., SHINN, G. A. t~ SANTOS, G. W. (1987). Cyclosporin A and the thymus. Immunopathology. Am. J. Path., 126, 487-496. BLACKMAN, M., KAPPLER, J. & MARRACK,P. (1990). The role of the T cell receptor in positive and negative selection of developing T cells. Science, 248, 1335- 1341. CLARKE, A. G. & KENDALL, M. D. (1989). Histological changes in the thymus during mouse pregnancy. Thymus, 14, 65 - 78. COrtEN, A., LEE, J. W. W., DOSCH, H.-M. & GELFAND, E. W. (1980). The expression of deoxyguanosine toxicity in T lymphocytes at different stages of maturation. J. Immun., 125, 1578- 1582. COOK, J. C., DOLD, K. M. & GREENLEE, W. F. (1987). An in vitro model for studying the toxicity of 2,3,7,8tetrachlorodibenzo-p-dioxin to human thymus. Toxic. appl. Pharmac., 89, 256-268. DARDENNE, M. & BACH, J. F. (1988). Functional biology of thymic hormones. Thymus Update, 1, 101 - 116. DUVALL, E. & WYLLIE, A. H. (1986). Death and the cell. Immun. Today, 4, 115-119. FINE, J. S., SILVERSTONE, A. E. & GASIEWlCZ, T. A. (1990). Impairment of prothymocyte activity by 2,3,7,8tetrachlorodibenzo-p-dioxin. J. Immun., 144, 1169- 1176. GLAZIER, A., TUTSCHKA,P. J., FARMER,E. R. & SANTOS,G. W. (1983). Graft-versus-host disease in cyclosporin A-treated rats after syngeneic and autologous bone marrow reconstitution. J. exp. Med., 158, 1 - 8. GREENLEE, W. F., DOLD, K. M., IRONS, R. D. & OSBORNE, R. (1985). Evidence for direct action of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) on thymic epithelium. Toxic. appl. Pharmac., 79, 112-120. GREENSTEIN, B. D., FITZPATRICK, F. T. A., ADCOCK, 1. M. & KENDALL,M. D. (1986). Reappearance of the thymus in old rats after orchidectomy: inhibition of regeneration by testosterone. J. Endocrinol., liD, 417- 422. GREENSTE1N, B. D., FITZPATRICK,F. T. A., KENDALL,M. D. & WHEELER, M. J. (1987). Regeneration of the thymus in old male rats treated with a stable analogue of LHRH. J. Endocrinol., 112, 345- 350. KENDALL,M. D. (1988). Anatomical and physiological factors influencing the thymic microenvironment. Thymus Update, 1, 27 - 65. KRAJNC, E. I., WESTER, P. W., LOEBER, J. G., VAN LEEUWEN, F. X. R., Vos, J. G., VAESSEN,H. A. M. G. & VAN DER HEYDEN, C. A. (1984). Toxicity of bis(tri-n-butyltin)oxide in the rat. 1. Short-term effects on general parameters and on the endocrine and lymphoid system. Toxic. appl. Pharmac., 75, 363- 384. LUSTER, M. I., BOORMAN, G. A., DEAN, J. H., HARRIS, M. W., LUEBKE, R. W., PADARATHSINGH,M. el al. (1980). Examination of bone marrow, immunologic parameters and host susceptibility following pre- and postnatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Int. J. Immunopharmac., 2, 301- 310. MA, D. D. F., SYLWESTROWICZ,T., JANOSSY, G. & HOFFBRAND,A. V. (1983). The role of purine metabolic enzymes and terminal deoxynucleotidyl transferase in intrathymic T-cell differentiation, lmmun. Today, 4, 6 5 - 68. MCCONKEY, D. J., HARTZELL, P., DUDDY, S. K., H~KANSSON,H. & ORRENIUS, S. (1988). 2,3,7,8-Tetrachlorodibenzo-pdioxin kills immature thymocytes by CaZ'-mediated endonuclease activation. Science, 242, 256-259. MCCONKEY, D. J., NICOTERA, P., HARTZELL, P., BELLOMO, G., WYLLIE, A. H. & ORRENIUS, S. (1989). Glucocorticosteroids activate a suicide process in thymocytes through an elevation of cytosolic Ca 2 concentrations. Arch. Biochem. Biophys., 269, 365- 370. McDONALD, H. R., LEES, R. K., BRON, C., SORDAT, B. & MIESCHER, G. H. (1987). T cell antigen receptor expression in athymic (nu/nu) mice. Evidence for an oligoclonal/3 chain repertoire. J. exp. Med., 166, 195-209. MURPHY, K. M., HEIMBERGER, A. B. & LOH, D. Y. (1990). Induction by antigen by intrathymic apoptosis of CD4 ~ CD8' TCR m thymocytes in vivo. Science, 250, 1720- 1722. PENNINKS, A. H., DIETERS, R. H. H., SNOEIJ, N. J. & SEINEN, W. (1991). Organotin-induced thymus atrophy. Thymus Update, 4, 5 7 - 79. PIETERS, R. H. H., KAMPINCIA, J., BoL-SCHOENMAKERS,M., LAM, B. W., PENNINKS, A. H. & SEINEN, W. (1989). Organotin-induced thymus atrophy concerns the OX-44 + immature thymocytes. Thymus, 14, 79-88. POLAND, A. & KNUTSON, J. C. (1982). 2,3,7,8-Tetrachlorodibenzo-p-dioxin and related halogenated hydrocarbons: examination of the mechanism of toxicity. A. Rev. Pharmac. Toxic., 22, 517-554. PUGH-HUMPHREVS, R. G. P., Ross, C. S. K. & THOMSON, A. W. (1990). The influence of FK-506 on the thymus. An immunophenotypic and structural analysis. Immunology, 70, 398- 404. SCHUURMAN, H.-J., DE WAAL, E. J., VAN LOVEREN, H. & VOS, J. G. (1991a). The toxicity of dioxin to the thymus. Thymus Update, 4, 3 5 - 55. VAN LOVEREN, H., SCHUURMAN,H.-J., KAMPINGA,J. &; Vos, J. G. Reversibility of thymic atrophy induced by 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) and bis(tri-n-butyltin)oxide (TBTO). Int. J. hnmunopharmac., 13, 369-377.
Chemicals Trophic for the Thymus
375
SCHUURMAN,H.-J, KRAJNC-FRANKEN,M. A. M., KUPER, C. F., VAN LOVEREN,H. & VOS, J. G. (1991b). Immune System. In Handbook o f Toxicologic Pathology (eds I-Iasheck-Hock, W. M. and Rosseaux, C. G.), pp. 421 -487. Academic Press, New York. SCHUURMAN,H.-J., NAGELKERKEN,L., DE WEGER, R. A. & ROZING, J. (1991c). Age-associated involution: significance of a physiologic process. Thymus, 18, 1 - 6 . SCHUURMAN,H. J., VAN LAARHOVEN,J. P. R. M., BROEKHU1ZEN,R., SPIERENBURG,G.Th., BREKELMANS,P., FIGDOR, C. G. & KATER, L. (1983). Lymphocyte maturation in the human thymus. Relevance of purine nucleotide metabolism for intrathymic T cell function. Scand. J. Immun., 18, 539-549. SCHUURMAN,H.-J., VAN LOVEREN,H., ROZlNG, J., VAN DIJK, A., LOEBER, J. G. & VOS, J. G. (1990). Cyclosporin and the rat thymus. An lmmunohistochemical study. Thymus, 16, 235- 254. SCOLLAV, R., BUTCHER, E. C. & WEISSMAN, I. L. (1980). Thymus cell migration. Quantitative aspects of cellular traffic from the thymus to the periphery in mice. Eur. J. lmmun., 10, 210-218. SCOLLAY,R., SMITH,J. • STAUFFER,V. (1986). Dynamics of early T cells: prothymocyte migration and proliferation in the adult mouse thymus, lmmun. Rev., 91, 129- 157. SELVE, H. (1936). Thymus and adrenals in the response of the organism to injuries and intoxications. Br. J. exp. Path., 17, 234. Sin, Y., SAHAI, B. M. & GREEN, D. R. (1989). Cyclosporin A inhibits activation-induced cell death in T-cell hybridomas and thymocytes. Nature, 339, 625- 626. SMITH, C. A., WILLIAMS, G. T., KINGSTON, R., JENKINSON, E. J. & OWEN, J. J. T. (1989). Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures. Nature, 337, 181 - 184. SNOE1J, N. J., PENNINKS,A. H. & SEINEN, W. (1988). Dibutyltin and tributyltin compounds induce thymus atrophy in rats due to a selective action on thymic lymphoblasts. Int. J. lmmunopharmac., 10, 891-899. SPRENT, J., GAO, E. K. & WEBB, S. R. (1990). T cell reactivity to MHC molecules: immunity versus tolerance. Science, 248, 1357- 1363. STEHR-GREEN, P. A., NAYLOR, P. H. & HOFFMAN, R. E. (1989). Diminished thymosino.~ levels in persons exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Toxic. Envir. Hlth, 28, 285-295. VAESSON, L. M. B., BROEKHUIZEN,R., ROZING, J., Vos, J. G. & SCHUURMAN,H.-J. (1986). T-cell development during ageing in congenitally athymic (nude) rats. Scand. J. lmmun., 24, 223- 235. VAN BAARLEN, J., SCHUURMAN,H.-J. & HUBER, J. (1988). Acute thymus involution in infancy and childhood: a reliable marker for duration of acute illness. Human Path., 19, 1155- 1160. VAN LOVEREN, H., SCHUURMAN,H.-J., KAMPINGA,J. & Vos, J. G. (1991). Reversibility of thymic atrophy induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and bis(tri-n-butyltin)oxide (TBTO). Int. J. Immunopharmac., 13, 369 - 377. YON BOEHMER, H. & KISIELOW, P. (1990). Self-nonself discrimination by T cells. Science, 248, 1369- 1372. Vos, J. G. & MOORE, J. A. (1974). Suppression of cellular immunity in rats and mice by maternal treatment with 2,3,7,8tetrachlorodibenzo-p-dioxin. Int. Arch. Allergy appl. Immun., 47, 777- 794.