Thymocyte migration: an affair of multiple cellular interactions?

Cell migration is a crucial event in the general process of thymocyte differentiation. The cellular interactions involved in the control of this migration are beginning to be defined. At least chemokines and extracellular matrix proteins appear to be part of the game. Cells of the thymic microenvironment produce these two groups of molecules, whereas developing thymocytes express the corresponding receptors. Moreover, although chemokines and extracellular matrix can drive thymocyte migration per se, a combined role for these molecules appears to contribute to the resulting migration patterns of thymocytes in their various stages of differentiation. The dynamics of chemokine and extracellular matrix production and degradation is not yet well understood. However, matrix metalloproteinases are likely to play a role in the breakdown of intrathymic extracellular matrix contents. Thus, the physiological migration of thymocytes should be envisioned as a resulting vector of multiple, simultaneous and/or sequential stimuli involving chemokines, adhesive and de-adhesive extracellular matrix proteins, as well as matrix metalloproteinases. Accordingly, it is conceivable that any pathological change in any of these loops may result in the alteration of normal thymocyte migration. This seems to be the case in murine infection by the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas' disease. A better knowledge of the physiological mechanisms governing thymocyte migration will provide new clues for designing therapeutic strategies targeting developing T cells

Characterization and distribution of extracellular matrix components and receptors in GH3B6 prolactin cells.

GH3B6 cells, a rat pituitary tumor cell line, synthesize and secrete large amounts of prolactin (PRL) in vitro. In the present work, we evaluated the capacity of these cells to express extracellular matrix (ECM) components and receptors in vitro. The expression of laminin (LN), fibronectin (FN) and type IV collagen (CIV) was investigated by immunofluorescence assays. In comparison to PRL distribution, where around 50-70% of the cells contained PRL concentrated in the Golgi region, a variable immunolabeling for the three ECM components could be observed in the majority of GH3B6 cells. Importantly, this pattern was not modified when cells were cultured in the presence of 30 nM thyroliberin (TRH). The expression of the ECM receptors: alpha5beta1 (FN receptor), alpha6beta1 (LN receptor) and CD44 (hyaluronic acid receptor) could be demonstrated by cytofluorometric analysis. Using biochemical procedures, we analyzed the synthesis and secretion of glycosaminoglycans (GAGs). The cells synthesized and secreted mainly heparan sulfate (75%) with a minor amount of chondroitin sulfate/dermatan sulfate. In an attempt to evaluate the individual contribution of the ECM components to influence cell morphology and PRL distribution in vitro, GH3B6 cells were cultivated separately on LN, FN and CIV substrates. Under all conditions, it was possible to observe an increase of cell adherence to the substrate, accompanied with changes of cellular morphology, characterized by the appearance of cytoplasmatic processes. However, no changes on PRL distribution could be observed. Our results suggest that endocrine tumor cell lines are involved in synthesis of ECM components and receptors.

Is there a glycosaminoglycan-related heterogeneity of the thymic epithelium?

We determined the synthesis and secretion of glycosaminoglycans by three distinct preparations of mouse cultured thymic epithelial cells. These comprised primary cultures of thymic nurse cells (TNCs), which are normally located within the cortex of the thymic lobules, as well as two murine thymic epithelial cells, bearing a mixed, yet distinct, cortico-medullary phenotype. We first identified and measured the relative proportions of the various glycosaminoglycans in the three epithelial cells. Non-sulfated glycosaminoglycans are preponderantly secreted by the TNCs, while the sulfated glycans (particularly heparan sulfate) are relatively more abundant on the cell surface. The three types of epithelial cells differ markedly in their heparan sulfate composition, mainly due to different patterns of N- and O-sulfation. In addition, the cells differ in the synthesis and secretion of other glycosaminoglycans. Thus, TNCs secrete high amounts of dermatan sulfate + chondroitin sulfate to the culture medium. IT-76M1 cells secrete high proportions of heparan sulfate while 2BH4 cells show a more equilibrated proportion of dermatan sulfate/chondroitin sulfate and heparan sulfate. The three epithelial cells also differ in their capacity to produce hyaluronic acid and 2BH4 cells are distinguished by their high rate of synthesis of this glycosaminoglycan. In conclusion, our results show that distinct thymic epithelial cells can synthesize different types of glycosaminoglycans. Although it remains to be definitely determined whether these differences reflect the in vivo situation, our data provide new clues for further understanding of how glycosaminoglycan-mediated interactions behave in the thymus.

The conveyor belt hypothesis for thymocyte migration: participation of adhesion and de-adhesion molecules.

Thymocyte differentiation is the process by which bone marrow-derived precursors enter the thymus, proliferate, rearrange the genes and express the corresponding T cell receptors, and undergo positive and/or negative selection, ultimately yielding mature T cells that will represent the so-called T cell repertoire. This process occurs in the context of cell migration, whose cellular and molecular basis is still poorly understood. Kinetic studies favor the idea that these cells leave the organ in an ordered pattern, as if they were moving on a conveyor belt. We have recently proposed that extracellular matrix glycoproteins, such as fibronectin, laminin and type IV collagen, among others, produced by non-lymphoid cells both in the cortex and in the medulla, would constitute a macromolecular arrangement allowing differentiating thymocytes to migrate. Here we discuss the participation of both molecules with adhesive and de-adhesive properties in the intrathymic T cell migration. Functional experiments demonstrated that galectin-3, a soluble beta-galactoside-binding lectin secreted by thymic microenvironmental cells, is a likely candidate for de-adhesion proteins by decreasing thymocyte interaction with the thymic microenvironment.

The conveyor belt hypothesis for thymocyte migration: participation of adhesion and de-adhesion molecules

Thymocyte differentiation is the process by which bone marrow-derived precursors enter the thymus, proliferate, rearrange the genes and express the corresponding T cell receptors, and undergo positive and/or negative selection, ultimately yielding mature T cells that will represent the so-called T cell repertoire. This process occurs in the context of cell migration, whose cellular and molecular basis is still poorly understood. Kinetic studies favor the idea that these cells leave the organ in an ordered pattern, as if they were moving on a conveyor belt. We have recently proposed that extracellular matrix glycoproteins, such as fibronectin, laminin and type IV collagen, among others, produced by non-lymphoid cells both in the cortex and in the medulla, would constitute a macromolecular arrangement allowing differentiating thymocytes to migrate. Here we discuss the participation of both molecules with adhesive and de-adhesive properties in the intrathymic T cell migration. Functional experiments demonstrated that galectin-3, a soluble ß-galactoside-binding lectin secreted by thymic microenvironmental cells, is a likely candidate for de-adhesion proteins by decreasing thymocyte interaction with the thymic microenvironment

Thymic epithelial cells synthesize a heparan sulfate with a highly sulfated region.

Epithelial cells are important components of the thymus microenvironment and are involved in thymocyte differentiation. The production and secretion of sulfated glycosaminoglycans by these cells grown in culture were investigated using labeling with radioactive 35S-Na2SO4 and 3H-glucosamine. The major glycosaminoglycans synthesized by these cells are heparan sulfate and hyaluronic acid. The structure of the heparan sulfate was investigated by the pattern of degradation products formed by deaminative cleavage with nitrous acid. The ratio 35S-sulfate/ H-glucosamine is high in the segments of the heparan sulfate released during the deaminative cleavage with nitrous acid but low in the resistant portion of the molecule. Thus, the heparan sulfate synthesized by the thymic epithelial cells contains a highly sulfated region. Digestion with heparitinase reveals that this highly sulfated region is a heparin-like segment of the molecule. The heparan sulfate is rapidly incorporated into the cell surface but its secretion to the extracellular medium requires a longer incubation period. Finally, heparin was used to mimic the possible effect of this heparan sulfate with a highly sulfated region, as ascertained by its ability to modulate thymocyte adhesion to thymic epithelial cells. Since heparin actually enhanced thymocyte adhesion, it is suggested that the heparan sulfate described herein, secreted by the thymic epithelium, may play a role upon intrathymic heterotypic cellular interactions.

Neuroendocrine control of the thymus.

Thymocytes undergo a complex process of differentiation, largely dependent on interactions with the thymic microenvironment, a tridimensional cellular network formed by epithelial cells, macrophages, dendritic cells, and fibroblasts. One key cellular interaction involves the TCR-CD3 complex expressed by thymocytes with MHC-peptide complexes present on microenvironmental cells. Additionally, thymic epithelial cells (TEC) interact with thymocytes via soluble polypeptides such as thymic hormones and interleukins, as well as through extracellular matrix (ECM) ligands and receptors. Such types of heterotypic interactions are under neuroendocrine control. For example, thymic endocrine function, represented by thymulin production, is up-regulated, both in vivo and in vitro, by thyroid and pituitary hormones, including prolactin and growth hormone. We also showed that these peptides enhance the expression of ECM ligands and receptors, as well as the degree of TEC-thymocyte adhesion. In addition, we studied the thymic nurse cell complex, used herein as an in vitro model for ECM-mediated intrathymic T-cell migration. We observed that T-cell migration is also hormonally regulated as ascertained by the thymocyte entrance into and exit from these lymphoepithelial complexes. Taken together these data clearly illustrate the concept that neuroendocrine circuits exert a pleiotropic control on thymus physiology. Lastly, the intrathymic production of classic hormones such as prolactin and growth hormone suggests that, in addition to endocrine circuits, paracrine and autocrine interactions mediated by these peptides and their respective receptors may exist in the thymus, thus influencing both lymphoid and microenvironmental compartments of the organ.

The thymic nurse cell complex: an in vitro model for extracellular matrix-mediated intrathymic T cell migration

The thymus is a primary lymphoid organ in wich bone narrow-derived T cell precursors undergo a complex maturation process in the context of the thymic microenvironment, represented by non-lymphoid cells and extracellular matrix (ECM) components. The thymic epithelial cells are the major cellular component of the thymic microenvironment, and influence different aspects of thymocyte differentiation, via cell-cell interactions and secretion of soluble factors, such as thymic hormones. The thymic nurse cell (TNC) complexes are multicellular lymphoepithelial structures formed by one thymic epithelial cell harboring 2-200 thymocytes, primary bearing the CD4/CD8 double-positive phenotype. TNCs probably create a special microenvironment for thymocyte differentiation and/or proliferation, with thymocytes being exposed to major histocompatibility complex (MHC) antigens and thymic hormones. Such differentiation parallels cell migration into and out of the complex. We showed the expression of ECM components and respective receptors by TNCs, and that interactions between the epithelial component of TNC and TNC-lymphocytes can be modulated by ECM components and respective receptors. Moreover, we demonstrated that intrinsic as well as extrinsic biological circuits can be involved in the control of such ECM-mediated thymic epithelial cell (TEC)/thymocyte interactions. For example, interferon-gamma can biphasically modulate the expression of ECM ligands and receptors by TEC, with results in corresponding modulation of their ability to interact with TNC-thymocytes. Additionally, hormones such as triiodothyronine, prolactin and growth hormone can influence the degree of these lymphocyte/epithelial cell adhesive interactions. Lastly, we recently furnished evidence for a de-adhesive mechanism within TNC aparently mediated by galectin 3 (an endogenous soluble beta-galactoside-binding lectin). Taken together, our data strongly indicate that thymic nurse cells can be regarded as an in vitro model for intrathymic T cell migration, particularly with respect to those events mediated by the extracellular matrix.

Carbohydrate-binding proteins in cell-matrix interactions.

1. Carbohydrate-dependent interactions have been more extensively studied during the last decade. Although the roles of carbohydrates in cellular functions are still poorly understood, the finding of carbohydrate-binding proteins in animal cells opened a great number of perspectives. 2. Animal lectins are associated with tumor progression, playing a key role in neoplastic cell interactions with endothelial cells and extracellular matrix glycoproteins such as laminin. 3. Here, we review the role of animal lectins in the migrating phenotype of neoplastic cells and normal cells such as T-lymphocytes.

Carbohydrate-binding proteins in cell-matrix interactions

1. Carbohydrate-dependent interactions have been more extensively studied during the last decade. Althought the roles of carbohydrates in cellular functions are still poorly understood, the finding of carbohydrate-binding proteins in animal cells opened a great number of perspectives. 2. Animal lectins are associated with tumor progression, playing a key role in neoplastic cell interactions with endothelial cells and extracellular matrix glycoproteins such as laminin. 3. Here, we review the role of animal lectins in the migrating phenotype of neoplastic cells and normal cells such as T-lymphocytes

Extracellular matrix components of the mouse thymic microenvironment. III. Thymic epithelial cells express the VLA6 complex that is involved in laminin-mediated interactions with thymocytes.

We describe herein the expression of the VLA6 complex by murine thymic epithelial cells (TEC). The immunohistochemical distribution revealed that VLA6 is found in both thymic medullary and subcapsullary areas. Moreover, studies by immunoelectron microscopy revealed a membrane labeling of the VLA6 molecule, including at desmosomal sites. By means of immunoblotting, immunoprecipitation, and affinity chromatography of extracts from a mouse TEC line, we further demonstrated that VLA6 is a laminin (LN) receptor in these cells. In keeping with this finding, we showed that TEC adhesion, spreading, and proliferation were enhanced in vitro by LN. The fact that VLA6 is also expressed by the large majority of thymocytes raised the hypothesis that it might be involved in LN-mediated TEC-thymocyte interactions. Interestingly, in vitro experiments showed that there is an increase in the TEC-thymocyte adhesion upon glucocorticoid hormone treatment, a situation in which the expression of VLA6 as well as LN is enhanced. Most importantly, this adhesion can be reversed by pre-treating TEC with an anti-alpha 6 integrin mAb. Additionally, spontaneous in vitro thymocyte release by thymic nurse cell complexes was enhanced by LN and partially blocked by anti-alpha 6 or anti-beta 1 antibodies. Our results suggest that VLA6 is involved in LN-mediated TEC-thymocyte interactions that can be relevant for thymic microenvironmental cell physiology and intrathymic T cell differentiation events.

Pleiotropic influence of triiodothyronine on thymus physiology.

It is well demonstrated that the thymus gland is under neuroendocrine control. Thymic endocrine function can be modulated by a variety of hormones including those secreted by the thyroid gland. This prompted us to investigate putative influences of T3 in further aspects of thymus physiology. We showed that T3-treated animals exhibited an increase in thymus weight, cellularity and cycling cells. Moreover, Thy1+ thymocytes as well as CD4-CD8 defined subsets were augmented in absolute numbers, whereas PgP.1+ cells increased in both absolute and percentage values. In parallel, the total numbers of thymic nurse cells were also increased. Regarding the expression of extracellular matrix components (ECM) by microenvironmental cells, we observed an enhancement in the intrathymic ECM upon T3 in vivo treatment. Similar effects were found in vitro by treating a thymic epithelial cell line or thymic nurse cell-derived epithelial cultures with T3. This treatment also increased the expression of ECM receptors by thymic epithelial cultures. Interestingly, an enhancement in thymocyte/thymic epithelial cell adhesion ratio was observed after T3 treatment of epithelial cells. Our data suggest that T3 exerts a pleiotropic effect upon thymus physiology, stimulating thymocyte differentiation, not only by modulating epithelial cell hormonal secretion but also their production of ECM proteins and respective receptors.

Extracellular matrix proteins in intrathymic T-cell migration and differentiation?

Intrathymic T-cell migration and differentiation is not completely understood. Here, Wilson Savino and colleagues argue that certain interactions between differentiating thymocytes and thymic epithelial cells are mediated by extracellular matrix proteins and that these interactions influence intrathymic migration events and thymocyte differentiation.

Identification of nuclear triiodothyronine receptors in the thymic epithelium.

Thymic epithelial cell physiology is known to be under neuroendocrine control. In particular, thyroid hormones modulate thymic hormone secretion by thymic epithelial cells in vivo and in vitro, thus suggesting the existence of specific receptors for those hormones in this component of the thymic microenvironment. Yet, thyroid hormone-binding sites have previously been detected only in crude thymus fractions and lymphocytes. We, thus, decided to search for T3 receptors in the thymic epithelium, by using an antinuclear T3 receptor monoclonal antibody. In situ immunohistochemical analysis of thymic frozen sections showed nuclear labeling of both lymphoid and nonlymphoid cells in the cortex and medulla. Moreover, in vitro studies using thymic epithelial cell lines and the so-called thymic nurse cells revealed a positive reaction in the chromatin, with nucleoli remaining negative. Immunoblot data clearly showed a single protein band of 57K reactive with the antinuclear T3 receptor antibody in murine thymus extracts as well as in the thymic epithelial cell lines. Lastly, in vitro treatment of these cells with T3 resulted in a transient, yet profound, down-modulation of the receptor. In conclusion, our findings provide molecular evidence that the action of thyroid hormones on thymic epithelium occurs via the typical 57K nuclear T3 receptors.