Déa Maria Serra Villa-Verde

Molecular mechanisms governing thymocyte migration: combined role of chemokines and extracellular matrix

Cell migration is crucial for thymocyte differentiation, and the cellular interactions involved now begin to be unraveled, with chemokines, extracellular matrix (ECM) proteins, and their corresponding receptors being relevant in such oriented movement of thymocytes. This notion derives from in vitro, ex vivo, and in vivo experimental data, including those obtained in genetically engineered and spontaneous mutant mice. Thymic microenvironmental cells produce both groups of molecules, whereas developing thymocytes express chemokine and ECM receptors. It is important that although chemokines and ECM proteins can drive thymocyte migration per se, a combined role of these molecules likely concurs for the resulting migration patterns of thymocytes in their various differentiation stages. In this respect, among ECM moieties, there are proteins with opposing functions, such as laminin or fibronectin versus galectin‐3, which promote, respectively, adhesion and de‐adhesion of thymocytes to the thymic microenvironment. How chemokines and ECM are produced and degraded remains to be more clearly defined. Nevertheless, matrix metalloproteinases (MMPs) likely play a role in the intrathymic ECM breakdown. It is interesting that these molecules also degrade chemokines. Thus, the physiological migration of thymocytes should be conceived as a resulting vector of multiple, simultaneous, or sequential stimuli, involving chemokines, adhesive, and de‐adhesive ECM proteins. Moreover, these interactions may be physiologically regulated in situ by matrix MMPs and are influenced by hormones. Accordingly, one can predict that pathological changes in any of these loops may result in abnormal thymocyte migration. This actually occurs in the murine infection by the protozoan Trypanosoma cruzi, the causative agent of Chagas disease. In this model, the abnormal release of immature thymocytes to peripheral lymphoid organs is correlated with the higher migratory response to ECM and chemokines. Lastly, the fine dissection of the mechanisms governing thymocyte migration will provide new clues for designing therapeutic strategies targeting developing T cells. The most important function of the thymus is to generate T lymphocytes, which once leaving the organ, are able to colonize specific regions of peripheral lymphoid organs, the T cell zones, where they can mount and regulate cell‐mediated, immune responses. This intrathymic T cell differentiation is a complex sequence of biological events, comprising cell proliferation, differential membrane protein expression, gene rearrangements, massive programmed cell death, and cell migration. In this review, we will focus on the mechanisms involved in controlling the migration of thymocytes, from the entrance of cell precursors into the organ to the exit of mature T cells toward peripheral lymphoid organs. Nevertheless, to better comprehend this issue, it appeared worthwhile to briefly comment on some key aspects of thymocyte differentiation and the tissue context in which it takes place, the thymic microenvironment.

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.

Control of human thymocyte migration by Neuropilin-1/Semaphorin-3A-mediated interactions.

It is largely established that molecules first discovered in the nervous system are also found in the immune system. Neuropilin-1 (NP-1) was initially identified to mediate semaphorin-induced chemorepulsion during brain development and is also involved in peripheral T cell/dendritic cell interactions. Herein, we studied NP-1 during T cell development in the human thymus. NP-1 is expressed in both cortex and medulla of thymic lobules, being found in distinct CD4/CD8-defined thymocyte subsets. NP-1 is also found in thymic epithelial cells (TEC) in situ and in vitro, and is recruited at the site of TEC–thymocyte contact. Moreover, NP-1 was rapidly up-regulated during thymocyte stimulation by T cell receptor (TCR) and IL-7 or after adhesion to TEC. Semaphorin-3A (Sema-3A), a natural ligand of NP-1, is also present in human thymus, both in TEC and thymocytes, being up-regulated in thymocytes after TCR engagement. Functionally, Sema-3A decreases the adhesion capacity of NP-1+ thymocytes and induces their migration by a repulsive effect. In conclusion, we show here that NP-1/Sema-3A-mediated interactions participate in the control of human thymocyte development.

Pituitary hormones modulate cell–cell interactions between thymocytes and thymic epithelial cells

The thymic microenvironment plays a key role in the intrathymic T-cell differentiation. It is composed of a tridimensional network of epithelial cells whose physiology is controlled by extrinsic circuits such as neuroendocrine axes. Herein we show that the expression of extracellular matrix ligands and receptor by cultured thymic epithelial cells is upregulated by prolactin (PRL) and growth hormone (GH), the latter apparently occurring via insulin-like growth factor I (IGF-I). Thymocyte release from the lymphoepithelial complexes, thymic nurse cells, as well as the reconstitution of these complexes are enhanced by PRL, GH or IGF-I. Treatment of a mouse thymic epithelial cell line with these hormones induced an increase in thymocyte adhesion, an effect significantly prevented in the presence of antibodies to fibronectin, laminin or respective receptors VLA-5 and VLA-6. Our data suggest that the in vitro changes in thymocyte/thymic epithelial cell interactions induced by pituitary hormones are partially mediated by the enhancement of extracellular matrix ligands and receptors.

Trypanosoma cruzi infection modulates intrathymic contents of extracellular matrix ligands and receptors and alters thymocyte migration

Several T cell abnormalities have been described in the course of acute Trypanosoma cruzi infection in mice, including severe effects on the thymus. In the present study, looking at the expression of extracellular matrix ligands in the thymus, we observed that deposits of fibronectin and laminin increased progressively during the course of infection, reaching a maximum at the peak of parasitemia and thymic atrophy. Concomitantly, membrane expression of fibronectin and laminin receptors (VLA‐4, VLA‐5 and VLA‐6) was also enhanced on thymocyte subsets of infected mice. These results correlated with changes in intrathymic thymocyte migration ability during the acute phase of infection, when a higher fibronectin‐dependent transmigratory activity of CD4+CD8+ thymocytes was observed. Strikingly, we detected higher frequency of immature and high VLA‐expressing CD4+CD8+ T cells in the peripheral lymphoid organs of infected mice at thepeak of parasitemia. These cells seemed to be thymus dependent, since significantly lower amounts of them were found in thymectomized mice, and some of them carry “prohibited” Vβ segments of the TCR. Our data suggest an imbalance in the intrathymic cell trafficking following acute T. cruzi infection, likely due to dysregulated extracellular matrix‐dependent interactions.

Functional Insulin-Like Growth Factor-1/Insulin-Like Growth Factor-1 Receptor-Mediated Circuit in Human and Murine Thymic Epithelial Cells

Interactions between thymocytes and thymic epithelial cell (TEC) can be modulated by growth hormone via insulin-like growth factor-1 (IGF-1). In this study, we showed IGF-1 and IGF-1 receptor mRNA expression by human and murine TEC and thymocytes. Functionally, IGF-1 stimulates extracellular matrix production by human TEC. Moreover, pretreatment of murine TEC with IGF-1 increases their adhesion to thymocytes. Interestingly, we observed an increase in the frequency of CD4–CD8–CD90+ T cells which adhered to pretreated TEC, supporting the concept that IGF-1 may also act indirectly on intrathymic T cell differentiation and migration through the thymic epithelium.

Differential Regional Immune Response in Chagas Disease

Following infection, lymphocytes expand exponentially and differentiate into effector cells to control infection and coordinate the multiple effector arms of the immune response. Soon after this expansion, the majority of antigen-specific lymphocytes die, thus keeping homeostasis, and a small pool of memory cells develops, providing long-term immunity to subsequent reinfection. The extent of infection and rate of pathogen clearance are thought to determine both the magnitude of cell expansion and the homeostatic contraction to a stable number of memory cells. This straight correlation between the kinetics of T cell response and the dynamics of lymphoid tissue cell numbers is a constant feature in acute infections yielded by pathogens that are cleared during the course of response. However, the regional dynamics of the immune response mounted against pathogens that are able to establish a persistent infection remain poorly understood. Herein we discuss the differential lymphocyte dynamics in distinct central and peripheral lymphoid organs following acute infection by Trypanosoma cruzi, the causative agent of Chagas disease. While the thymus and mesenteric lymph nodes undergo a severe atrophy with massive lymphocyte depletion, the spleen and subcutaneous lymph nodes expand due to T and B cell activation/proliferation. These events are regulated by cytokines, as well as parasite-derived moieties. In this regard, identifying the molecular mechanisms underlying regional lymphocyte dynamics secondary to T. cruzi infection may hopefully contribute to the design of novel immune intervention strategies to control pathology in this infection.

Triiodothyronine Modulates Extracellular Matrix-Mediated Interactions between Thymocytes and Thymic Microenvironmental Cells

Objectives: Thyroid hormones exert immunomodulatory activities and the thymus is one of their target organs. We previously showed that triiodothyronine (T3) modulates thymic hormone production and extracellular matrix (ECM) expression by mouse thymic epithelial cells (TEC). This concept is enlarged herein by studying the effects of T3 in human TEC preparations including primary cultures derived from thymic nurse cell complexes, as well as human and murine TEC lines. Methods and Results: We observed that in all cases, ECM ligands and receptors (such as fibronectin, laminin, VLA-5 and VLA-6) are enhanced in vitro, as ascertained by immunocytochemistry, ELISA and cytofluorometry. Moreover, thymocyte adhesion to these TEC preparations is augmented by T3. Interestingly, TEC-thymocyte adhesion is also upregulated when thymocytes from T3-treated mice adhere to untreated TEC cultures. Such an enhancing effect of T3 upon TEC-thymocyte interactions is likely due to the increase in the expression of ECM ligands and receptors, since it is prevented when T3-treated TEC cultures are incubated with anti-ECM antibodies prior to the adhesion assay. We then tested whether T3 could modulate interactions between thymocytes and nonepithelial microenvironmental cells, exemplified herein by the phagocytic cells of the mouse thymic reticulum. In fact, in vitro treatment of these cells with T3 increases ECM ligands and receptors and augments their ability to adhere to thymocytes. Lastly, using immunochemistry-based assays, we showed the presence of the nuclear T3 receptor in all thymic microenvironmental cell preparations. Conclusion: Our data show that T3 upregulates ECM-mediated heterocellular interactions of thymocytes with distinct thymic microenvironmental cells, in both humans and mice.

Impaired migration of NOD mouse thymocytes: a fibronectin receptor-related defect.

We previously showed intrathymic alterations in non‐obese diabetic (NOD) mice, including the appearance of giant perivascular spaces, filled with mature thymocytes, intermingled with an extracellular matrix network. This raised the hypothesis of a defect in thymocyte migration with partial arrest of exiting thymocytes in the perivascular spaces. Herein, we investigated the expression of receptors for fibronectin [very late antigen (VLA)‐4 and VLA‐5] and laminin (VLA‐6), known to play a role in thymocyte migration. When compared with two normal and one other autoimmune mouse strains, a decrease of VLA‐5 expression in NOD thymocytes was noticed, being firstly observed in late CD4/CD8 double‐negative cells, and more pronounced in mature CD4+ and CD8+ thymocytes. Functionally, thymocyte exit from the lymphoepithelial complexes, the thymic nurse cells, was reduced. Moreover, NOD thymocyte adhesion to thymic epithelial cells as well as to fibronectin was diminished, and so was the migration of NOD thymocytes through fibronectin‐containing transwell chambers. In situ, intra‐perivascular space thymocytes were VLA‐5‐negative, suggesting a correlation between the thymocyte arrest within these structures and loss of VLA‐5 expression. Overall, our data reveal impairment in NOD thymocyte migration, and correspond to the first demonstration of a functional fibronectin receptor defect in the immune system.

Caspase-8 and caspase-9 mediate thymocyte apoptosis in Trypanosoma cruzi acutely infected mice

Trypanosoma cruzi acute infection leads to thymic atrophy, largely as a result of death of immature DP T cells. In a second vein, the glucocorticoid hormone imbalance promotes DP T cell apoptosis in infected mice. Herein, we assessed the involvement of caspase signaling in thymocyte death during T. cruzi acute infection. BALB/c mice were infected i.p. with 102 trypomastigote forms of T. cruzi and analyzed from 7 to 19 dpi. Thymocyte apoptosis was observed in early stages of infection, increasing along with time postinfection. Immature DN and DP as well as CD4+ and CD8+ thymocytes from infected mice showed increased activation of caspase‐8, ‐9, and ‐3. In vitro treatment of thymocytes from infected mice with a general caspase inhibitor or the combination of caspase‐8‐ and caspase‐9‐specific inhibitors increased the number of living thymocytes. Intrathymic injection of the general caspase inhibitor, but not caspase‐8 or ‐9 inhibitors individually, prevented thymic atrophy and thymocyte depletion in infected mice. Moreover, blockade of glucocorticoid receptor activity with RU486 prevented DP thymocyte apoptosis, together with caspase‐8 and ‐9 activation. These findings indicate that DP T cell apoptosis following experimental T. cruzi acute infection is dependent on glucocorticoid stimulation, promoting caspase‐8 and ‐9 activation.