Indomethacin inhibits thymic involution in mice with streptozotocin-induced diabetes

Diabetes is chronic disease that is accompanied by a rapid thymus involution. To investigate the factors responsible for thymic involution in a model of STZ-induced diabetes, mice were injected with STZ alone or in combination with the cyclooxygenase 2 inhibitor indomethacin (INDO). Thymus weight, glycemia and serum corticosterone were measured, and apoptosis in thymus and thymocyte cultures was analyzed by flow cytometry. Although earlier studies report that streptozotocin (STZ) is toxic to lymphoid tissues, in our experiments even massive doses of STZ did not negatively affect thymocyte cultures. Cultured thymocytes also seemed unaffected by high glucose concentrations, even after 24 h of exposure. Administration of INDO concomitantly with STZ reduced thymic involution but did not prevent the onset of hyperglycemia or reduce established hyperglycemia. When INDO was given before STZ, the same degree of thymic involution occurred; however, hyperglycemia was reduced, although normoglycemia was not restored. INDO also reduced serum corticosterone. Because thymocytes are known to be sensitive to glucocorticoids, this finding suggests that cyclooxygenase 2 inhibition may retard thymic involution by reducing serum glucocorticoids. In conclusion, our results show that STZ and hyperglycemia are not toxic to thymocytes and that cyclooxygenase 2-mediated mechanisms are involved in thymic involution during diabetes.     

Failure of rearranged TCR transgenes to prevent age-associated thymic involution.

After puberty, the thymus undergoes a dramatic loss in volume, in weight and in the number of thymocytes, a phenomenon termed age-associated thymic involution. Recently, it was reported that age-associated thymic involution did not occur in mice expressing a rearranged transgenic (Tg) TCRalphabeta receptor. This finding implied that an age-associated defect in TCR rearrangement was the major, if not the only, cause for thymic involution. Here, we examined thymic involution in three other widely used MHC class I-restricted TCRalphabeta Tg mouse strains and compared it with that in non-Tg mice. In all three TCRalphabeta Tg strains, as in control mice, thymocyte numbers were reduced by approximately 90% between 2 and 24 mo of age. The presence or absence of the selecting MHC molecules did not alter this age-associated cell loss. Our results indicate that the expression of a rearranged TCR alone cannot, by itself, prevent thymic involution. Consequently, other presently unknown factors must also contribute to this phenomenon.     

The human thymus: A new perspective on thymic function,aging, and hiv infection

Shortly after birth, the human thymus begins a life long process of involution, whereby the net size of the thymus is not altered but the organ is replaced by adipose tissue. As a result, it has long been believed that thymic involution is indicative of a nonfunctional organ. Recently, however, with the use of computed tomography analysis and innovative molecular approaches that measure T-cell receptor circles, indicative of recent thymic emigrants, doubt has been placed on that dogma. The thymus appears to be active in thymopoiesis throughout the adult life, albeit inversely correlated with age. Being faced with diseases that deplete T-cells such as the acquired immunodeficiency syndrome (AIDS), this recent finding has the potential to exploit novel approaches that enhance thymic output as a mechanism to reconstitute the immune system. In this review, we will revisit the role of T-cells in immunity, the relationship between thymic function and age, and closely examine the impact of HIV-mediated thymic dysregulation on thymopoiesis.     

Lysosomal membrane-associated glycoproteins are differentially expressed in acute and chronic human thymic involution

The lysosomal membrane-associated glycoproteins LAMP-1 and LAMP-2 are the major constituents of lysosomal membranes with still undefined biological functions. As autophagy is an alternative model of programmed cell death in which lysosomes play a crucial role, we hypothesize that LAMPs might participate in this phenomenon in the involuting thymus. Thymic glands from cases with acute (infection induced) and chronic (senile) involution were examined immunohistochemically for the expression of LAMPs. In acute involution LAMP-1 was localized mainly in medullary epithelial cells, in single macrophages and lymphocytes. Hassall's corpuscules were stained less intensely as compared to control specimens. The quantitative analysis showed a significantly elevated LAMP-2 expression compared to LAMP-1. LAMPs were detected with very slight reactivity in the senile thymus. The enhanced expression of LAMPs, and mainly of LAMP-2, in epithelial cells of incidentally involuted thymus might be an indicator of acute cell injury requiring autophagic degradation of damaged structures. The diminished expression of LAMPs in age-involuted thymus could be a sign of the morphological reorganization and the functional disregulation of the gland. In conclusion, we present novel evidence for differential expression of LAMP-1 and LAMP-2 in thymic involution suggesting their possible involvement in the process of accidental involution of the thymic gland.     

Occurrence of GABA-transaminase in the thymus gland of juvenile and aged rats

The occurrence and distribution of GABA-transaminase (GABA-t) activity were examined in the thymus of juvenile, adult and aged rats, using enzyme-histochemical and biochemical methods. Quantitative image analysis showed that specific GABA-t reactivity was localized in the wall of the arteries and, to a lesser extent, to the veins. Only a low activity could be observed in association with the subcapsular and medullary part of the parenchyma of the thymus. Many structures resembling nerve fibers also showed low positive reactivity. Biochemical results gave the following decreasing order of GABA-t activity: arteries, veins, whole thymus and parenchyma. Histoenzymatic staining and related values of quantitative analysis of images, are in agreement with the biochemical results; moreover, they demonstrated that the intensity of histoenzymatic staining for GABA-t in thymus of rats strongly decreases with age. GABA-t in thymic tissue is concentrated in blood vessels and particularly in the arteries. Therefore, our findings do not support the earlier assumptions that GABA-t is exclusively concentrated in cerebral vessels. Moreover, the decrease of GABA-t activity during age in thymic tissues may be related to the decrease of thymic microvessels as consequence of the thymic involution. On the contrary, the thymic macrovessels show an elevated staining in all ages with an apparent increase due to the thymic involution.     

Are zinc-bound metallothionein isoforms (I+II and III) involved in impaired thymulin production and thymic involution during ageing?

Background  

With advancing age, thymic efficiency shows progressive decline due to thymic involution allowing impaired cell-mediated immunity and the appearance of age-related diseases. The intrinsic cause of thymic involution is still undefined. Chronic inflammation and high glucocorticoids (GCs) may be involved. However, transgenic mice, with increased GC sensitivity and over expression of GC receptors, display delayed age-associated thymic involution. This fact suggests that other substances may affect thymic involution. Among them, both isoforms of metallothioneins (MTs) I+II and III are the major candidates because their increments leads to organ atrophy in constant stress and are induced by IL-6, which increases in ageing. Enhanced MTs in ageing allows constant sequester of zinc ions and no subsequent zinc release leading to low zinc ion bioavailability for thymic efficiency. This sequester is very limited in very old age. Thus, we have investigated the MTmRNA (I+II and III) in the thymus from young, old and very old mice.     

Quantification of thymic innervation in juvenile and aged rats.

Thymic innervation has been quantified using silver impregnation and light microscopy on 5 juvenile and 5 aged rats. There is an increase in innervation density of the thymus with age, exceeding the reduction of thymic volume due to involution. The density of innervation correlates inversely with thymic volume. From previous studies, we conclude that thymic innervation is confined to the extraparenchymal compartment. Ultrastructural morphometry shows this compartment to maintain its volume during involution of the remaining thymic tissue. Related only to the extraparenchymal compartment, the innervation increases by a factor two with age.     

The initial age-associated decline in early T-cell progenitors reflects fewer pre-thymic progenitors and altered signals in the bone marrow and thymus microenvironments

Age-related thymus involution results in decreased T-cell production, contributing to increased susceptibility to pathogens and reduced vaccine responsiveness. Elucidating mechanisms underlying thymus involution will inform strategies to restore thymopoiesis with age. The thymus is colonized by circulating bone marrow (BM)-derived thymus seeding progenitors (TSPs) that differentiate into early T-cell progenitors (ETPs). We find that ETP cellularity declines as early as 3 months (3MO) of age in mice. This initial ETP reduction could reflect changes in thymic stromal niches and/or pre-thymic progenitors. Using a multicongenic progenitor transfer approach, we demonstrate that the number of functional TSP/ETP niches does not diminish with age. Instead, the number of pre-thymic lymphoid progenitors in the BM and blood is substantially reduced by 3MO, although their intrinsic ability to seed and differentiate in the thymus is maintained. Additionally, Notch signaling in BM lymphoid progenitors and in ETPs diminishes by 3MO, suggesting reduced niche quality in the BM and thymus contribute to the early decline in ETPs. Together, these findings indicate that diminished BM lymphopoiesis and thymic stromal support contribute to an initial reduction in ETPs in young adulthood, setting the stage for progressive age-associated thymus involution.     

Lymphohematopoietic progenitors do not have a synchronized defect with age-related thymic involution

It has been speculated that aging lymphohematopoietic progenitor cells (LPC) including hematopoietic stem cells (HSC) and early T-cell progenitors (ETP) have intrinsic defects that trigger age-related thymic involution. However, using a different approach, we suggest that that is not the case. We provided a young thymic microenvironment to aged mice by transplanting a fetal thymus into the kidney capsule of aged animals, and demonstrated that old mouse-derived LPCs could re-establish normal thymic lymphopoiesis and all thymocyte subpopulations, including ETPs, double negative subsets, double positive, and CD4(+) and CD8(+) single positive T cells. LPCs derived from aged mice could turn over young RAG(-/-) thymic architecture by interactions, as well as elevate percentage of peripheral CD4(+)IL-2(+) T cells in response to costimulator in aged mice. Conversely, intrathymic injection of ETPs sorted from young animals into old mice did not restore normal thymic lymphopoiesis, implying that a shortage and/or defect of ETPs in aged thymus do not account for age-related thymic involution. Together, our findings suggest that the underlying cause of age-related thymic involution results primarily from changes in the thymic microenvironment, causing extrinsic, rather than intrinsic, defects in T-lymphocyte progenitors.     

Effects of T-activin on the morphology of the thymus gland in experimental injury of the lower limbs in mice (anti-stress effect of T-activin)

The effect of T-activin on thymic involution under the experimental trauma of femur was studied. T-activin in a dose of 1.0 micrograms/mouse was injected into young male (CBA X C57BL)F1 mice weighing 17.5-19.0 g before (I injection) or immediately after the fracture of the femur during 3 days. Morphometric analysis of the thymus was made 1, 5, 10 and 15 days after the trauma. It was found that T-activin suppressed the involution of the thymus, induced by the trauma, during the first 5 days and accelerate the process of its regeneration. It is suggested, that T-activin displays protective anti-stress effect on the mouse thymic involution.     

Immunoendocrine modulation and stimulation of hematopoiesis with the ionophore copolymer T150R1

T150R1, an 8000-dalton copolymer with sodium ionophore activity, has been shown to modulate cellular responses in multiple systems. In this article, we studied its effects on lymphoid and hematopoietic organs in the context of the adrenal-pituitary axis. When injected in mice as an oil in water emulsion, T150R1 caused a rapid, profound, and dose-dependent thymic involution accompanied by splenic hyperplasia. Time course experiments with a 2.5-mg dose revealed that the thymus size was minimal at Day 2, rose to normal by Day 14, then enlarged and gradually returned to normal by Week 6 postinjection. Thymic involution was due to cellular depletion of the cortical area, whereas thymic enlargement was due to cortical hyperplasia. Splenomegaly was seen as early as Day 4, peaked by Day 14, and gradually returned to normal by Week 6. The splenic enlargement was due to hyperplasia of the red pulp, with evidence of proliferating erythropoietic, myelopoietic, and megakaryopoietic precursors. In addition, the bone marrow was stimulated and extramedullary hematopoiesis was present in the liver. The effects of T150R1 on the thymus appeared to be mediated by corticosteroids while the effects on hematopoiesis were not. Corticosterone and ACTH levels were increased in treated animals. Adrenalectomy diminished the T150R1-induced thymic involution but enhanced the splenic hyperplasia. Hypophysectomy did not prevent thymic involution, suggesting that T150R1 has endocrine stimulatory effects. These data suggest that T150R1 represents a new class of ionophores which may act on excitable cells within the endocrine, immune, and hematopoietic systems.     

The effects of age, thymectomy, and HIV Infection on alpha and beta TCR excision circles in naive T cells

Due to homeostasis total naive T cell numbers remain fairly constant over life despite a gradual involution of the thymus. The contribution of the thymus to maintaining naive T cell pools is typically measured with TCR excision circles (TRECs) that are formed in thymocytes. The mechanisms underlying thymic involution are poorly understood. Some data suggest that thymocytes undergo fewer divisions in old (small) than young (large) thymi, and other data suggest that the number of TRECs per thymocyte is independent of age. If thymic involution were associated with a decreased number of divisions of the thymocytes, this would markedly complicate the interpretation of TREC data. To study this we develop a mathematical model in which the division rate of thymocytes decreases with increasing age. We describe the dilution of TRECs formed during the arrangement of both chains of the TCR by division of thymocytes, recent thymic emigrants, and mature naive T cells. The model behavior is complicated as TREC contents in naive T cells can increase with age due to decreased dilution in the thymus. Because our model is consistent with current data on the effects of age and thymectomy on TRECs in peripheral T cells, we conclude that aging may well affect thymocyte division, which markedly complicates the interpretation of TREC data. It is possible, but more difficult, to let the model be consistent with the rapid changes in alpha and beta TRECs observed shortly after HIV infection.     

Age-related involution and terminal disorganization of the human thymus

The terminal involution pattern of the human thymus was studied based on autopsy cases (both sexes, age range 63-91 years). Large sections through the entire thymic fat body were examined with the help of both conventional histological and immunohistochemical techniques. The findings demonstrate that thymic atrophy in old humans (a) goes far beyond the degree of involution observed in small rodents; (b) results in a system of thin, branching, in part interrupted, non-keratinizing epithelial plates containing no typical Hassall bodies; (c) concerns all components of the thymus except fat tissue which progressively replaces original thymic structures; and (d) involves various types of disorganization of individual lobules with T and B lymphocytes often located outside rather than within epithelial remnants. Effects of low-level radiation on this final regression of the human thymus are unknown.     

Expression of nerve growth factor is upregulated in the rat thymic epithelial cells during thymus regeneration following acute thymic involution

Neuroimmune networks in the thymic microenvironment are thought to be involved in the regulation of T cell development. Nerve growth factor (NGF) is increasingly recognized as a potent immunomodulator, promoting "cross-talk" between various types of immune system cells. The present study describes the expression of NGF during thymus regeneration following acute involution induced by cyclophosphamide in the rat. Immunohistochemical stain demonstrated not only the presence of NGF but also its upregulated expression mainly in the subcapsular, paraseptal, and perivascular epithelial cells, and medullary epithelial cells including Hassall's corpuscles in both the normal and regenerating thymus. Biochemical data obtained using Western blot and RT-PCR supported these results and showed that thymic extracts contain NGF protein and mRNA, at higher levels during thymus regeneration. Thus, our results suggest that NGF expressed in these thymic epithelial cells plays a role in the T lymphopoiesis associated with thymus regeneration during recovery from acute thymic involution.     

Declining expression of a single epithelial cell‐autonomous gene accelerates age‐related thymic involution

Age‐related thymic involution may be triggered by gene expression changes in lymphohematopoietic and/or nonhematopoietic thymic epithelial cells (TECs). The role of epithelial cell‐autonomous gene FoxN1 may be involved in the process, but it is still a puzzle because of the shortage of evidence from gradual loss‐of‐function and exogenous gain‐of‐function studies. Using our recently generated loxP‐floxed‐FoxN1(fx) mouse carrying the ubiquitous CreER^T (uCreER^T) transgene with a low dose of spontaneous activation, which causes gradual FoxN1 deletion with age, we found that the uCreER^T‐fx/fx mice showed an accelerated age‐related thymic involution owing to progressive loss of FoxN1⁺ TECs. The thymic aging phenotypes were clearly observable as early as at 3–6 months of age, resembling the naturally aged (18–22‐month‐old) murine thymus. By intrathymically supplying aged wild‐type mice with exogenous FoxN1‐cDNA, thymic involution and defective peripheral CD4⁺ T‐cell function could be partially rescued. The results support the notion that decline of a single epithelial cell‐autonomous gene FoxN1 levels with age causes primary deterioration in TECs followed by impairment of the total postnatal thymic microenvironment, and potentially triggers age‐related thymic involution in mice.     

Effects of castration on thymocyte development in two different models of thymic involution

Age-associated thymic involution is accompanied by decreased thymic output. This adversely affects general immune competence and T cell recovery following cytoreductive treatments such as chemotherapy. A causal link between increasing sex steroids and age-related thymic atrophy is well established. Although castration has been demonstrated to regenerate the atrophied thymus, little is known about how this is initiated or the kinetics of thymocyte regeneration. The present study shows that although castration impacts globally across thymocyte development in middle-aged mice, the regenerative effects are initiated in the immature triple-negative compartment and early T lineage progenitors (ETP). Specifically, there was a reduction in number of ETP with age, which was restored following castration. There was, however, no change in ETP reconstitution potential in ETP at this age or following castration. Furthermore, in a chemotherapy-induced model of thymic involution, we demonstrate castration enhances intrathymic proliferation and promotes differentiation through the triple-negative program. Clinically, reversible sex steroid ablation is achieved hormonally, and thus presents a means of ameliorating immune inadequacies, for example, following chemotherapy for bone marrow transplantation. By improving our understanding of the kinetics of thymic recovery, this study will allow more appropriate timing of therapy to achieve maximal reconstitution, especially in the elderly.     

Implants of quail thymic epithelium generate permanent tolerance in embryonically constructed quail/chick chimeras

In situ implantation of a quail wing bud into a chick embryo at 4 days of incubation (E4) regularly results in the normal development of the implant followed by its acute rejection starting within two weeks post-hatching. If the epithelial thymic rudiments of the quail donor are implanted into the branchial arch area of the chick recipient after partial removal of its own thymic primordia, a chimeric thymus develops in the chick host and this induces tolerance to the quail wing by the chick recipient. The species identity of cells in chimeric thymuses was mapped using Feulgen-Rossenbeck' staining and immunolabelling with monoclonal antibodies directed against quail or chick B-L antigens. Certain lobes contained only chick cells both at the stromal and hemopoietic cell levels. Others had a quail epithelial stroma containing host hemopoietically derived cells. Only chimeras in which at least one third of the thymic lobes were chimeric showed permanent tolerance to the grafted wing. Since the two species exhibit distinct developmental rates, we decided to study the kinetics of thymic involution after birth. Although the changes in thymus weight and histological structure are fundamentally similar in quail and chick, those in the quail start about 7-8 weeks earlier. In the chimeric thymuses, the lobes whose epithelial cells were quail involuted at the rate of control quail showing no influence of the hemopoietic thymic compartment in this process. Tolerance induced by the thymic epithelium during embryogenesis and in early postnatal life was maintained after a profound involution of the quail thymic graft had occurred.     

Effect of age on T cell differentiation.

A brief overview of the area of T cell aging is presented by first discussing the age-related changes in T cell activities, and then by focusing attention on the possible mechanisms that may be responsible for the decline. Present evidence indicates that thymic involution precedes and therefore may be responsible for the age-dependent decline in the ability of the immune system to generate functional T cells. At this time, it appears that the primary effect of thymic involution is on a T cell differentiation pathway affecting the more mature T cells first with time, and then the less mature T cells. Thus, the thymus may be the aging clock for the immune system. Further studies should be centered around processes regulating growth and atrophy of the thymus.     

Up-regulation of VEGF expression by NGF that enhances reparative angiogenesis during thymic regeneration in adult rat

Angiogenesis is important for adult tissue regeneration as well as normal development. Vascular endothelial growth factor (VEGF) is a unique potent angiogenic factor, and plays an essential role in regulating angiogenesis during embryonic development, normal tissue growth, and tissue regeneration. Recent evidence shows that nerve growth factor (NGF) also plays a role as an angiogenic regulator as well as a well-known neurotrophic factor. The aim of this study was to investigate whether thymus regeneration accompanies reparative angiogenesis and also to evaluate whether the thymic expression of VEGF is regulated by NGF in vivo and in vitro. Here, we show that high VEGF mRNA and protein levels are concomitant with reparative angiogenesis that occurs dramatically during regeneration following acute involution induced by cyclophosphamide (CY) in the rat thymus. Fluorescent thymus angiography using FITC-dextran showed that thymic regeneration is associated with a much denser capillary network compared with normal control thymus. Furthermore, the expressions of NGF and TrkA were highly increased during thymic regeneration. We also show that NGF mediates thymic epithelial induction of VEGF expression in vitro and in vivo. Taken together, our results suggest that NGF-mediated VEGF up-regulation in thymic epithelial cells may contribute to reparative angiogenesis during thymic regeneration in adult.