Lysosomal-mediated degradation of apoptotic thymocytes within thymic nurse cells

A thymic epithelial cell line (tsTNC-1) that maintains the ability to selectively bind and internalize immature alphabetaTCR(lo)CD4(+)CD8(+) thymocytes in vitro was used in long-term coincubation experiments to determine the ultimate fate of thymocytes that remained within intracytoplasmic vacuoles of thymic nurse cells (TNCs). In an earlier report, a subset of the population released from the TNC interaction was shown to mature to the alphabetaTCR(hi)CD69(hi) stage of development, while thymocytes that bided within the TNC cytoplasm died through the process of apoptosis. Here, we show the presence of both apoptotic and nonapoptotic thymocytes within the cytoplasm of freshly isolated TNCs as well as in tsTNC-1 cells in culture. A microscopic analysis revealed total degradation of the cytoplasmic apoptotic thymocyte population that remained in tsTNC-1 cells after an 8- to 10-h incubation period. A quantitative analysis showed an increase of cytoplasmic thymocyte degradation over time to almost 80% after 9 h of incubation. However, in the presence of bafilomycin A1, which is used to inhibit acidification of lysosomal vesicles, degradation of apoptotic thymocytes never reached 10%. These data suggest that lysosomes within TNCs play a role in the degradation of apoptotic thymocytes. We examined tsTNC-1 cells before the addition of thymocytes to cultures and found lysosomes to be clustered around the nucleus in the cytoplasm of TNCs. Shortly after the internalization event, apoptotic thymocytes move to the area of the cytoplasm containing lysosomes. Using the confocal microscope, we obtained evidence that shows the degradation event to be facilitated through the fusion of lysosomes with the specialized vacuoles within TNCs containing apoptotic cells.     

Thymic nurse cells exclusively bind and internalize CD4+CD8+ thymocytes.

Thymic nurse cells (TNC) contain 20-200 thymocytes within specialized vacuoles in their cytoplasm. The purpose of the uptake of thymocytes by TNCs is unknown. TNCs also have the capacity to present self-antigens, which implies that they may serve a function in the process of thymic education. We have recently reported the development of thymic nurse cell lines that have the ability to bind and internalize T cells. Here, we use one of these TNC lines to identify the thymocyte subpopulation(s) involved in this internalization process. TNCs exposed to freshly isolated thymocytes bind and internalize CD4 and CD8 expressing thymocytes (CD4+CD8+ or double positives) exclusively. More specifically, a subset of the double-positive thymocyte population displayed binding capacity. These double-positive cells express cell surface alpha beta type T cell antigen receptor (TCR), as well as CD3 epsilon. Binding was not inhibited in the presence of antibodies against CD3, CD4, CD8, Class I antigens, or Class II antigens. These results describe two significant events in T cell development. First, TNCs exclusively bind and internalize a subset of alpha beta TCR expressing double-positive T cells. Also, binding is facilitated through a mechanism other than TCR recognition of major histocompatibility complex antigens. This suggests that thymocyte internalization may be independent of the process used by TNCs to present self-antigen.     

Circulating macrophages as well as developing thymocytes are enclosed within thymic nurse cells.

Both thymic nurse cells (TNCs) and macrophages have been reported to function as antigen-presenting cells during the process of MHC restriction. Negative selection, which results in the apoptosis of potentially autoreactive thymocytes, is believed to be associated with both macrophages and TNCs in the cortex. Both cell types have also been reported to ingest thymocytes undergoing positive and negative selection. However, macrophages ingest apoptotic thymocytes, while TNCs have been shown to internalize viable cells. A subset of the TNC-engulfed population is allowed to mature and is released, while the remaining fraction becomes apoptotic and is absorbed within the TNC cytoplasm through lysosomal activity. A recent report described a subset of rat TNCs that contain macrophages as well as thymocytes within their cytoplasm. We examined freshly isolated TNCs from C57BL/6 mice and found that, of the TNC population recovered, 1.7% contained macrophages within its cytoplasm. There also were macrophages tightly bound but not internalized into the multicellular structure at a rate of 2.9%. The total association of macrophages with TNCs was approximately 4.6%. This unique association of macrophages with TNCs was also observed in vitro when freshly isolated thymocytes (containing macrophages) were added to cultures of cells from the TNC cell line tsTNC-1. The macrophage-TNC interaction was found to be dynamic, with macrophages moving rapidly into and out of TNCs containing cytoplasmic thymocytes. Macrophages within TNCs showed a close association with cytoplasmic thymocytes. We then labeled peritoneal macrophages with CFDA SE, a cell tracking dye, and returned them to the mouse peritoneum. Within 1 h, labeled macrophages were detectable in the thymus. This is the first investigation to show a direct interaction between peripheral macrophages and TNCs. These results suggest that TNCs and macrophages work together as antigen-presenting cells.     

Lymphocyte depletion in thymic nurse cells: a tool to identify in situ lympho-epithelial complexes having thymic nurse cell characteristics

Summary In situ pre-existing complexes of epithelial cells and thymocytes having thymic nurse cell characteristics were visualized in the murine thymus cortex using dexamethasone as a potent killer of cortisone-sensitive thymocytes. The degradation and subsequent depletion of cortisone-sensitive thymocytes enclosed within cortical epithelial cells appeared to be paralleled by thymocyte degradation and depletion in thymic nurse cells isolated from thymic tissue fragments from dexamethasone-treated animals. This suggests that thymic nurse cells are derived from pre-existing sealed complexes of cortical epithelial cells and thymocytes. Not all thymocytes situated within in situ epithelial or thymic nurse cells complexes appear to be cortisone-sensitive: a minority of 1–2 thymocytes per complex survives the dexamethasone-treatment, thus constituting a minor subset of cortical cortisone-resistant thymocytes predominantly localized within cortical epithelial cells in situ and within thymic nurse cells derived from such structures. Cortisone resistance in thymocytes thus seems to be acquired within the cortical epithelial cell microenvironment. Cortisone-resistant thymocytes in thymic nurse cells express the phenotype of mature precursors of the T helper lineage, indicating that the in situ correlates of thymic nurse cells may play an important role in T cell maturation and selection.     

Thymic nurse cell multicellular complexes in HY-TCR transgenic mice demonstrate their association with MHC restriction

This study examines thymic nurse cell (TNC) function during T-cell development. It has been suggested that TNCs function in the removal of nonfunctional and/or apoptotic thymocytes and do not participate in major histocompatibility complex restriction. We analyzed TNCs isolated from both normal C57BL/6 mice and C57BL/6 TgN (TCRHY) mice (HY-TCR transgenic mice). Using confocal microscopic analyses of TNCs isolated from C57BL/6 animals, we showed that 75%-78% of the enclosed thymocyte subset was viable, and 87%-90% of these cells expressed both CD4 and CD8. CD4 and CD8 also were expressed on TNC thymocytes isolated from both male and female HY-TCR transgenic mice. The transgenic female thymus was shown to have 17 times more TNCs per milligram of thymus than the transgenic male thymus. TNCs from HY-TCR transgenic females were 8-10 microm larger than transgenic male TNCs, and the female TNCs contained five times more thymocytes within intracytoplasmic vacuoles, with less than 4% apoptosis. However, more than 42% of the thymocytes within transgenic male TNCs were apoptotic. The large number and size of TNCs containing viable thymocytes in the female transgenic thymus suggest that TNC function is not limited to the removal of apoptotic thymocytes. We believe that the selective uptake of viable double-positive thymocytes by TNCs in C57BL/6 and HY-TCR transgenic female mice provides evidence that this interaction occurs during the process of major histocompatibility complex restriction.     

Questionable thymic nurse cell.

Since their discovery in 1980, thymic nurse cells (TNCs) have been controversial. Questions pertaining to the existence of the TNC as a "unit" cell with thymocytes completely enclosed within its cytoplasm were the focus of initial debates. Early skeptics proposed the multicellular complex to be an artifact of the procedures used to isolate TNCs from the thymus. Since that time, TNCs have been found in fish, frogs, tadpoles, chickens, sheep, pigs, rats, mice, and humans. Their evolutionary conservation throughout the animal kingdom relieved most speculations about the existence of TNCs and at the same time demonstrated their apparent importance to the thymus and T-cell development. In this review we will discuss and debate reports that describe (i) the organization or structure of TNCs, (ii) the thymocyte subset(s) found within the cytoplasm of TNCs and their uptake and release, and (iii) the function of this fascinating multicellular interaction that occurs during the process of T-cell development. Discussions about the future of the field and experimental approaches that will lead to answers to remaining questions are also presented.     

Fatty acid-binding protein 4 (FABP4) and FABP5 modulate cytokine production in the mouse thymic epithelial cells

Thymic stromal cells, including cortical thymic epithelial cells (cTEC) produce many humoral factors, such as cytokines and eicosanoids to modulate thymocyte homeostasis, thereby regulating the peripheral immune responses. In this study, we identified fatty acid-binding protein (FABP4), an intracellular fatty acid chaperone, in the mouse thymus, and examined its role in the control of cytokine production in comparison with FABP5. By immunofluorescent staining, FABP4(+) cells enclosing the thymocytes were scattered throughout the thymic cortex with a spatial difference from the FABP5(+) cell that were distributed widely throughout the cTEC. The FABP4(+) cells were immunopositive for MHC class II, NLDC145 and cytokeratin 8, and were identified as part of cTEC. The FABP4(+) cells were identified as thymic nurse cells (TNC), a subpopulation of cTEC, by their active phagocytosis of apoptotic thymocytes. Furthermore, FABP4 expression was confirmed in the isolated TNC at the gene and protein levels. To explore the function of FABP in TNC, TSt-4/DLL1 cells stably expressing either FABP4 or FABP5 were established and the gene expressions of various cytokines were examined. The gene expression of interleukin (IL)-7 and IL-18 was increased both in FABP4 and FABP5 over-expressing cells compared with controls, and moreover, the increase in their expressions by adding of stearic acids was significantly enhanced in the FABP4 over-expressing cells. These data suggest that both FABPs are involved in the maintenance of T lymphocyte homeostasis through the modulation of cytokine production, which is possibly regulated by cellular fatty acid-mediated signaling in TEC, including TNC.     

Questionable Thymic Nurse Cell

Since their discovery in 1980, thymic nurse cells (TNCs) have been controversial. Questions pertaining to the existence of the TNC as a “unit” cell with thymocytes completely enclosed within its cytoplasm were the focus of initial debates. Early skeptics proposed the multicellular complex to be an artifact of the procedures used to isolate TNCs from the thymus. Since that time, TNCs have been found in fish, frogs, tadpoles, chickens, sheep, pigs, rats, mice, and humans. Their evolutionary conservation throughout the animal kingdom relieved most speculations about the existence of TNCs and at the same time demonstrated their apparent importance to the thymus and T-cell development. In this review we will discuss and debate reports that describe (i) the organization or structure of TNCs, (ii) the thymocyte subset(s) found within the cytoplasm of TNCs and their uptake and release, and (iii) the function of this fascinating multicellular interaction that occurs during the process of T-cell development. Discussions about the future of the field and experimental approaches that will lead to answers to remaining questions are also presented.     

Immunohistochemical characterization of nurse cells in normal human thymus.

Lymphoepithelial complexes known as thymic "nurse" cells (TNC) have been isolated and described in the thymus of several animal species including man. Most of the investigations on TNC have been carried out in enzymatically digested thymuses in which TNC were isolated by differential sedimentation. In the present study we demonstrate TNC in immunohistochemically stained sections of human thymus as ring-shaped cells completely enclosing thymocytes and localized not only in the cortex, but also at the corticomedullary junction where they have not been previously described. TNC expressed epithelial markers [low and high molecular weight keratins identified by 35 beta H11 and 34 beta E12 monoclonal antibodies, a cortical antigen shared with neuroectodermal neoplasms recognized by the GE2 monoclonal antibody, and tissue polypeptide antigen (TPA:B1)], class II histocompatibility antigens (HLA-DR), and thymosin alpha 1. Double staining experiments with the nuclear proliferation-associated antigen Ki-67 and the cortical epithelium marker GE2 showed that most thymocytes enclosed in these cortical TNC were not proliferating. The antigens expressed by TNC indicate that not only cortical, but also medullary epithelial cells are part of the TNC system. The possible role of TNC in the education and maturation of thymocytes is discussed.     

Production of prostaglandin E2 and prostacyclin by thymic nurse cells in culture

To better define the thymic microenvironment, we have examined a specific population of thymic stromal cells, thymic nurse cells (TNC) for production of eicosanoids. TNC were prepared from BALB/c mice, cultured in complete medium, and culture supernatants were analyzed for the presence of various metabolites of arachidonic acid. Freshly isolated TNC produced large quantities of PGE2 and 6-keto PGF1 alpha (a stable metabolite of prostacyclin, PGI2). Both of these eicosanoids were produced continuously in culture, after an initial lag period of approximately 2 h. No significant production of the eicosanoids PGD2, thromboxane B2, or leukotrienes B4, C4/D4/E4 was seen in these cultures. Production of PGE2 and PGI2 by TNC was not stimulated by treatment with the calcium ionophore A23187, or by cell-cell interactions resulting from coculture of the TNC with free thymocytes. Eicosanoid production in these cultures was not due to production of these substances by cells likely to be present as contaminants, such as T rosettes or free thymocytes. These findings raise the possibility that PGE2 and/or PGI2 may provide signals to thymocytes at a specific developmental stage.     

Stromal cell networks regulate thymocyte migration and dendritic cell behavior in the thymus

After entry into thymus, T cell progenitors migrate in the cortex and the medulla while completing their education. Recent reports have documented the dynamic and tortuous behavior of thymocytes. However, other than chemokines and/or segregated thymic substrates, the factors contributing to the dynamic patterns of thymocyte movement are poorly characterized. By combining confocal and dynamic two-photon microscopy, we demonstrate that thymocytes continuously migrate on thymic stromal cell networks. In addition to constituting "roads" for thymocytes, we observed that these networks also provide a scaffold on which dendritic cells attach themselves. These results highlight the central role of stromal microanatomy in orchestrating the multiple cellular interactions necessary for T cell migration/development within the thymus.     

Immunohistology of thymic nurse cells

The demonstration of thymic nurse cells (TNC), complexes between stromal cells and thymocytes, in cell suspensions of murine thymuses, prompted us to investigate (1) the relationship of TNC to other thymic stromal cell types defined in situ, and (2) the maturation stage of the enclosed thymocytes. To this purpose we incubated frozen sections of TNC suspensions with various monoclonal antisera directed to T cells and stromal cell types, using immunohistology. This approach enabled us to study antigen expression on the "nursing" cell itself and to analyze the phenotype of the enclosed lymphocytes in cross sections of TNC. The results show that lymphocytes enveloped by TNC express high levels of Thy-1, moderate levels of T200, and variable amounts of Lyt-1. Due to enzymatic degradation Lyt-2 expression could not be studied. The enveloped cells also bear PNA receptors, but no detectable I-A/E antigens. Expression of H-2K antigens on enclosed thymocytes varied from weak to absent. The "nursing" cells react with ER-TR4, a monoclonal antibody which detects cortical epithelial-reticular cells. In addition TNC express I-A/E and H-2K antigens. In contrast, TNC do not react with ER-TR 5 and 7, monoclonal antibodies, which detect medullary epithelial cells and reticular fibroblasts, respectively. TNC do not express the macrophage antigens Mac-1 and Mac-2. We conclude that TNC in vitro represent the in vivo association of epithelial-reticular cells with cortical thymocytes. However, the enclosed thymocytes do not constitute a phenotypically distinct subset of subcapsular or outer cortical cells.     

Identification and in situ localization of the "thymic nurse cell" in man

The observation of the "thymic nurse cell" (TNC), a reticuloepithelial cell with intracytoplasmic lymphocytes, in suspension of murine thymic tissue prompted us to investigate the existence of this cell in cell suspension, as well as in tissue sections of the human thymus. TNC-like cells were enriched in suspension by enzymatic disintegration of thymic tissue and 1 X G sedimentation over 50% fetal calf serum gradients. TNC-like cells were negative for lysosomal enzymes: in this respect, as well as in light microscopic morphology, the cells were different from tissue macrophages with intracytoplasmic lymphocytes. In electron microscopy, TNC-like cells showed reticuloepithelial characteristics. In 1-micron tissue sections, clusters of lymphocytes with a possible reticuloepithelial nucleus were observed close to blood capillaries in the cortical area. Ultrastructural analysis confirmed the epithelial nature of this cell, as well as its location adjacent to blood capillaries. We concluded that there is in situ existence of TNC in man. This observation enables studies on the role of TNC in intrathymic T cell maturation.     

Effect of cytokines on thymic hematopoietic precursors

Summary We have previously shown that the interaction of thymocytes with thymic accessory cells (macrophages and/or interdigitating cells) is one of the factors required for thymocyte activation. Precursors of both thymic accessory cell and thymocytes are included in the CD4^- CD8^- Mac-1^- Ia^- subpopulation, and their respective maturation and/or activation may be modulated by granulocyte-macrophage colony-stimulating factor, interleukin 1 and interleukin 2. When CD4^- CD8^- thymic cells are activated with granulocyte-macrophage colony-stimulating factor plus interleukin 2, both macrophages and interdigitating-like cells are present, as shown by electron microscopy. When activated with interleukin 1 plus interleukin 2, the interdigitating-like cells is the only accessory cell present. In both culture conditions, large clusters are formed between interdigitating cells and lymphoid cells. These results have led us to propose two-step signals for thymocyte proliferation: first, the maturation of macrophages under granulocyte-macrophage colony-stimulating factor control and the production of interleukin 1, and secondly, the maturation of interdigitating cells under interleukin 1 control, their clustering with thymocytes which are then activated.Abbreviations CFU-S colony-forming units in the spleen - CSF colony-stimulating factor - DC dendritic cells - DN double negative cells (CD4^- CD8^-) - EC epithelial cells - GM-CFC granulocyte/macrophage colony-forming cells - GM-CSF granulocytemacrophage CSF - IDC interdigitating cell - IL-1 interleukin 1 - IL-2 interleukin 2 - MØ macrophage - P-TR phagocytic cell of the thymic reticulum     

Expression and Distribution of the Adrenomedullin System in Newborn Human Thymus

Adrenomedullin (AM) is a multifunctional peptide endowed with various biological actions mediated by the interaction with the calcitonin receptor-like receptor (CLR), which couples to the receptor activity-modifying proteins 2 or 3 (RAMP2 or RAMP3) to form the functional plasma membrane receptors AM1 and AM2, respectively. In this study, we investigated for the first time the expression and localization of AM, CLR, RAMP2 and RAMP3 in human thymic tissue from newborns and in primary cultures of thymic epithelial cells (TECs) and thymocytes. Immunohistochemical analysis of thymic tissue showed that both AM and RAMP2 are abundantly expressed in the epithelial cells of medulla and cortex, blood vessels and mastocytes. In contrast, RAMP3 could not be detected. In cultured TECs, double immunofluorescence coupled to confocal microscopy revealed that AM is present in the cytoplasmic compartment, whereas RAMP2 could be detected in the cytoplasm and nucleus, but not in the cell membrane. At variance with RAMP2, CLR was not only present in the nucleus and cytoplasm of TECs, but could also be detected in the cell membrane. The nuclear and cytoplasmic localizations of RAMP2 and CLR and the absence of RAMP2 in the cell membrane were confirmed by western-blot analysis performed on cell fractions. AM, RAMP2 and CLR could also be detected in thymocytes by means of double immunofluorescence coupled to confocal microscopy, although these proteins were not present in the whole thymocyte population. In these cells, AM and RAMP2 were detected in the cytoplasm, whereas CLR could be observed in the cytoplasm and the plasma membrane. In conclusion, our results show that the AM system is widely expressed in human thymus from newborns and suggest that both AM1 receptor components CLR and RAMP2 are not associated with the plasma membrane of TECs and thymocytes but are located intracellularly, notably in the nucleus.     

The ER-TR4 monoclonal antibody recognizes murine thymic epithelial cells (Type 1) and inhibits their capacity to interact with immature thymocytes: immuno-electron microscopic and functional studies

The thymic stroma is heterogeneous with regard to cellular morphology and cellular function. In this study, we employed the monoclonal antibody ER-TR4 to characterize stromal cells at the ultrastructural level. To identify the labelled cell type, we used two techniques: immunogold labelling on ultrathin frozen sections and immunoperoxidase staining on thick vibratome sections. ER-TR4 reacted with thymic Type 1 epithelial cells (according to our classification). A dense labelling appears in the cytoplasm of cortical cells using the two techniques. Immunogold labelling identified small cytoplasmic vesicles whereas the cytoplasm and the cell membrane seem to be labelled with the immunoperoxidase technique. ER-TR4 also identified isolated thymic nurse cells (TNC), and was observed in vitro to inhibit the capacity of some type 1 epithelial cells to establish interactions with immature thymocytes. This finding supports the hypothesis that the factor is involved in the formation of lymphoepithelial interactions within thymic nurse cells, and thus in the relations that immature thymocytes establish with the thymic microenvironment.     

Thymic nurse cells: division of thymocytes within complexes

Summary Thymic nurse cell complexes (TNC-c), isolated from mouse thymuses at 1 and 2 h after i.v. injection of 6-(³H)thymidine, were analyzed in autoradiographs of semithin serial sections with regard to their size and the distribution of labeled thymocytes in individual types of complexes. The total number of thymocytes per complex reflects the type of complex. In a parallel study, localization of labeled thymocytes within individual zones of thymic cortex was examined. Thymocyte division within complexes may yield sequential complex generations differing in number per complex. However, thymocytes within complexes differ from each other in division kinetics. Half of the thymocytes that had been labeled 1 h after injection divided within 2 h. The rapidly dividing fraction of thymocytes were distributed within small complexes containing 2–8 cells and corresponded to the distribution of labeled cells in the outer thymic cortex. The proportion of labeled cells within large complexes resembled the distribution of labeled cells in the deep cortex. The data support the view that microenvironmental factors within TNC-c are responsible for both inducing thymocytes to enter the cell cycle and the negative selection (cell death) of some thymocytes.