Proteínas Hedgehog: expresión y función en el timo / Hedgehog proteins: expression and function in the thymus

Evidencias crecientes demuestran que los morfógenos clásicos, altamente conservados durante la evolución y tradicionalmente implicados en el desarrollo embrionario, donde determinan diferenciación celular y patrones de desarrollo, son expresados en tejidos adultos, como son la médula ósea y el sistema inmunitario. Estas moléculas podrían ser piezas importantes del rompecabezas que orquesta la diferenciación y la homeostasis del sistema inmune, aunque sea sólo recientemente cuando estamos comenzando a entender donde encajan en el entramado del sistema inmune. En esta revisión, describimos la ruta de señalización de las proteínas Hedgehog (Hh) centrándonos en su implicación en la diferenciación de las células T y su posible conexión con otros morfógenos, como son las proteínas morfogénicas del hueso (Bone Morphogenetic Proteins, BMPs) (AU)

Distinct mechanisms contribute to generate and change the CD4:CD8 cell ratio during thymus development: a role for the Notch ligand, Jagged1.

In adult life, the high CD4:CD8 cell ratio observed in peripheral lymphoid organs originates in the thymus. Our results show that the low peripheral CD4:CD8 cell ratio seen during fetal life also has an intrathymic origin. This distinct production of CD4(+)CD8(-) and CD4(-)CD8(+) thymocytes is regulated by the developmental age of the thymic stroma. The differential expression of Notch receptors and their ligands, especially Jagged1, throughout thymus development plays a key role in the generation of the different CD4:CD8 cell ratios. We also show that the intrathymic CD4:CD8 cell ratio sharply changes from fetal to adult values around birth. Differences in the proliferation and emigration rates of the mature thymocyte subsets contribute to this change.

Analysis of the human neonatal thymus: evidence for a transient thymic involution.

The neonatal period is marked by the impairment of the major components of both innate and adaptive immunity. We report a severe depletion of cortical CD4+CD8+ double-positive thymocytes in the human neonatal thymus. This drastic reduction in immature double-positive cells, largely provoked by an increased rate of cell death, could be observed as early as 1 day after birth, delaying the recovery of the normal proportion of this thymocyte subset until the end of the first month of postnatal life. Serum cortisol levels were not increased in newborn donors, indicating that the neonatal thymic involution is a physiological rather than a stress-associated pathological event occurring in the perinatal period. Newborn thymuses also showed increased proportions of both primitive CD34+CD1- precursor cells and mature TCRalphabetahighCD69-CD1-CD45RO+/RAdull and CD45ROdull/RA+ cells, which presumably correspond to recirculating T lymphocytes into the thymus. A notable reinforcement of the subcapsular epithelial cell layer as well as an increase in the intralobular extracellular matrix network accompanied modifications in the thymocyte population. Additionally neonatal thymic dendritic cells were found to be more effective than dendritic cells isolated from children's thymuses at stimulating proliferative responses in allogeneic T cells. All these findings can account for several alterations affecting the peripheral pool of T lymphocytes in the perinatal period.

Role of glucocorticoids in early T-cell differentiation.

The results of the T-cell differentiation in the progeny of adrenalectomized pregnant rats (Adx fetuses), an experimental model that ensures the absence of glucocorticoids (GCs) during the first stages of development, are summarized. In Adx thymuses there is an accelerated maturation of thymocytes that is reversed by in vivo GC replacement. In addition, Adx thymuses show decreased cell content, which correlates with both the increased numbers of apoptotic cells and an early migration of DP (CD4+CD8+) and SP (both CD4+CD8- and CD4-CD8+) thymocytes to the spleen. As shown by in vitro recolonization assays, accelerated T-cell differentiation is a consequence of changes in the biology of lymphoid precursors occurring in the fetal liver of Adx fetuses. They arrive at the thymic primordium earlier and mature faster than the fetal liver lymphoid progenitors from Sham control fetuses. After the establishment of a fetal hypothalamus-pituitary-gland-adrenal-gland (HPA) axis, there is a gradual normalization of the T-cell development Adx fetuses.

Early maturation of T-cell progenitors in the absence of glucocorticoids.

In the present work, we demonstrated that both fetal liver and thymic T-cell precursors express glucocorticoid receptors (GRs) indirectly suggesting a role for glucocorticoids (GCs) in the earliest events of T-cell differentiation. To evaluate this issue, we analyzed the thymic ontogeny in the progeny of adrenalectomized pregnant rats (Adx fetuses), an in vivo experimental model, which ensures the absence of circulating GCs until the establishment of the fetal hypothalamus-pituitary-adrenal (HPA) axis. In the absence of maternal GCs, T-cell development was significantly accelerated, the process being reversed by in vivo GC replacement. Mature single positive thymocytes (both CD4 and CD8) appeared in 16-day old fetal Adx thymus when in the control fetuses, most thymocytes still remained in the double-negative (DN) CD4(-)CD8(-) cell compartment. In addition, emigration of T-cell receptor (TcR)alphabeta positive cells to the spleen also occurred earlier in Adx fetuses than in control ones. In vitro recolonization of cultured deoxiguanosine-treated mouse fetal thymus lobes with 13-day-old fetal liver cell suspensions from both Adx and control fetuses demonstrated changes in the developmental capabilities of fetal liver T-cell precursors from embryos grown in the absence of GCs. Furthermore, a precocious lymphoid colonization of the thymic primordium from Adx fetuses was evidenced by ultrastructural analysis of both Adx and Sham early thymus. Both findings accounted for the accelerated T-cell differentiation observed in Adx fetuses. Together, these results support a role for GCs not only in the thymic cell death, but also in the early steps of T-cell differentiation.

Partial blockade of T-cell differentiation during ontogeny and marked alterations of the thymic microenvironment in transgenic mice with impaired glucocorticoid receptor function.

Glucocorticoids (GCs) are widely known to be potent modulators of the immune system. The role of GCs in thymopoiesis as well as the integration of the thymus with the neuroendocrine system is, however, poorly understood. In the present work, we have studied, in transgenic mice with an impaired GC function, the alterations which occur in both T-cell differentiation and thymic stroma maturation, throughout ontogeny as well as in adult condition, analyzing their possible rebounding on the status of adult splenic T lymphocyte populations. These transgenic mice have been described to present a significant decrease (60-70%) of thymic and splenic GC receptor binding capacity but maintain normal their basal plasma ACTH and corticosterone levels. The animals showed a partial blockade of T-cell differentiation and decreased percentages of apoptotic cells during fetal development but not in adult life, when thymic cellularity was significantly increased although thymocyte apoptosis response was not affected. In contrast, thymic stroma was profoundly altered from early fetal stages and large epithelium-free areas appeared in adult thymus. On the other hand, our study revealed a reduction of the splenic TcRalphabeta population accompanied by an increase in the CD4/CD8 ratio. The analysis of different adhesion molecules as well as activation markers demonstrated that most of them (CD5, CD11a, CD11b, CD69 and MHC Class II) were normally expressed in transgenic lymphocytes, whereas CD44 and CD62L expression was altered indicating the existence of an increased proportion of primed T-cells in these animals. In view of the mutual interdependence of thymic stroma and thymocyte maturation, the partial blockade of T-cell differentiation during ontogeny and the profound alterations of the stromal cell compartment in transgenic mice with impaired GR function suggest a key role for GCs in coordinating the physiological dialogue between the developing thymocytes and their microenvironment.

Glucocorticoid-mediated regulation of thymic dendritic cell function.

The possible effects of glucocorticoids (GC) on the biology of thymic dendritic cells (DC) have been analyzed. Both DC and GC seem to be involved in intrathymic T cell selection but possible relationships, if any, between them remain currently unknown. For the first time, we have proved the expression of GC receptors in thymic DC. Moreover, our data demonstrate that in vitro dexamethasone (Dex) treatment barely affects the viability of mature thymic DC, which are largely resistant to its apoptotic effect. Dex-treated thymic DC also show a slightly reduced surface expression of some adhesion and co-stimulatory molecules in correlation with diminished allostimulatory properties. Furthermore, the production of both IL-1beta and tumor necrosis factor (TNF)-alpha, but not that of IL-6 and IL-10, diminished in the mixed leukocyte reaction established with Dex-treated thymic DC. However, the addition of recombinant rat IL-1beta and TNF-alpha, alone or in combination, did not recover the allostimulatory capacity. Taken together, these results support certain GC-mediated regulation of the activity of thymic DC which could be relevant for the biology of the thymus gland.

Early differentiation of thymic dendritic cells in the absence of glucocorticoids.

The possible role of glucocorticoids (GCs) in the maturation of thymic dendritic cells (DCs) during early ontogeny was analyzed in the progeny of adrenalectomized pregnant rats (Adx foetuses). This experimental model ensured the lack of GCs until establishment of foetal hypothalamus-pituitary gland-adrenal (HPA) axis, and showed profound modifications of the development of thymus gland. In the absence of maternal GCs, there was a high percentage of DCs, many of them exhibiting a mature phenotype, in the 15-16 day-old Adx foetal thymus, which sharply decreased to reach control values on foetal day 17. On the other hand, the absolute number of DCs of Sham foetal rats increased throughout ontogeny, whereas the high numbers found in 15-16 day-old Adx foetuses significantly diminished in the following days. This process was closely correlated with the thymocyte life span, previously demonstrated, and the early appearance of DCs in the spleen. Our results demonstrate that like for other cell components of rat thymus, DC maturation is accelerated in an early foetal microenvironment devoid of glucocorticoids.

Accelerated maturation of the thymic stroma in the progeny of adrenalectomized pregnant rats.

The possible role played by glucocorticoids (GCs) in the development of thymic stromal cell components has been studied in the progeny of adrenalectomized pregnant rats (FAdx), an experimental model which ensures the absence of GCs until the establishment of the fetal hypothalamus-pituitary gland-adrenal gland axis. As previously demonstrated for thymocytes, the lack of GCs early in ontogeny results in an accelerated maturation of the thymic stromal elements. Early expression of specific cell markers for thymic epithelial cell subsets and appearance of a well-established cytokeratin-positive epithelial cytoreticulum confirmed the ultrastructural evidence of a faster maturation of the thymic epithelium in FAdx than in FSham. A similar faster and stronger pattern of both class I and class II molecule expression on the epithelial cells occurred in the former fetuses than in control ones. Changes in the pattern of expression of laminin, but not that of fibronectin, throughout thymic maturation also reflected accelerated maturation. Immunohistochemically identified thymic macrophages appeared late in both FSham and FAdx but in higher numbers in these latter indirectly demonstrating their faster development. Finally, the maturation and turnover of thymic dendritic cells showed a remarkable acceleration in the FAdx. In 15- to 16-day-old FAdx thymuses there was a high number of dendritic cells which sharply decreased in the following days suggesting a massive migration to the periphery and/or in situ cell death. In parallel a new wave of dendritic cell progenitors began to differentiate in the FAdx thymuses but not in the FSham ones. The results are discussed from the view of close relationships known to occur between thymocytes and the stromal components, although a direct effect of GCs cannot be discarded.

Interleukin-7 influences the development of thymic dendritic cells.

Interleukin-7 (IL-7) has been shown to be a critical factor in B and T lymphopoiesis, and to influence the differentiation of myeloid cell lineages. In the present study we extend these results demonstrating that IL-7 also plays an important role in the development of thymic dendritic cells (DC). The addition of IL-7 to rat fetal thymus organ cultures (FTOC) resulted in a drastic increase in the number of CD3(-)CD4(-)CD8(-) cells, which mostly expressed typical DC markers, including major histocompatibility complex class II, OX-62, CD11b, CD68, and CD54. These cells exhibited morphological and ultrastructural features of DC, and were potent stimulators of the allogeneic mixed leukocyte reaction. Although increased numbers of DC were continuously generated throughout the culture period in the presence of IL-7, they were not actively dividing, indicating that DC in IL-7-treated cultures did not arise by expansion of pre-existing cells. Reduced DC numbers obtained after the addition of neutralizing anti-IL-7 antibodies to mouse FTOC confirmed the relevance of endogenously produced IL-7 on thymic DC development. Furthermore, the addition of IL-7 to FTOC derived from severe combined immunodeficient mice also generated large numbers of DC in the absence of thymocyte maturation.

Expression of functionally active alpha 4 beta 1 integrin by thymic epithelial cells.

We have investigated the expression and function of the VLA-4 heterodimer alpha 4 beta 1, a member of the beta 1 integrin subfamily, on human thymic epithelial cells (TEC) derived from cortical epithelium. The expression of the alpha 4 integrin chain was studied in four different cloned TEC lines derived from either fetal or post-natal human thymus by both flow cytometry and immunoprecipitation techniques with anti-alpha 4 MoAbs. All different cell lines assayed expressed significant levels of alpha 4, as revealed by their reactivity with MoAbs specific for distinct alpha 4 epitopes. The alpha 4 subunit expressed by TEC was associated to beta 1 but not to beta 1 chain, and displayed the characteristic 80/ 70 kD pattern of proteolytic cleavage. The VLA-4 integrin in these cells was constitutively active in terms of adhesiveness to both fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In addition, this heterodimer localized to punctate regions of the cell in the area of contact with the substratum, named point contacts assessed by staining with the anti-beta 1 activation epitope 15/7 MoAb. According to the cortical origin of the TEC lines expressing VLA-4, human thymus sections stained with different anti-alpha 4 antibodies revealed the presence of cortical, and in smaller numbers medullary epithelial cells bearing alpha 4 integrin. The expression of alpha 4 in the thymus was also found in both adult and fetal rats, in which epithelial cells were also specifically stained. Altogether, our data show that VLA-4 is an additional component of the integrin repertoire of TEC, and suggest that it could have an important role in thymus epithelial cell-thymocyte interactions.

Histogenesis of the epithelial component of rat thymus: an ultrastructural and immunohistological analysis.

BACKGROUND: Despite the assumed importance of thymic cell microenvironments for governing T-cell maturation, little is known about the ontogeny of their cell components. A few studies have analyzed previously the ontogenetical development of rat thymic epithelium (Bogojevic et al. 1990. Period. Biol., 92:126; Kampinga and Aspinall 1990 Harwood Acad. Pub., London, pp. 149-186; Micic et al., 1991 Dev. Comp. Immunol., 15:443-450) and recently we have reported the development of both interdigitating/dendritic cells and macrophages (Vicente et al., 1994 Immunology, 82:75-81, 1995 Immunology, 85:99-105). METHODS: In the present work we analyze in situ ultrastructural, immunohistochemical, and histoenzymatically the appearance and development of the thymic epithelial cell component in both embryonic and neonatal Wistar rats with special emphasis on the origin of the different epithelial cell types, the occurrence or absence of a common precursor for these, and the expression of MHC molecules. RESULTS: The thymic primordium of 13-day-old embryos is formed by a homogeneous population of primitive epithelial cells differentiating gradually into various epithelial cell subtypes of both the cortex and the medulla. In the cortex, subcapsular and stroma-supporting epithelial cells appear at days 14-15 as two structurally different cell entities. At the same time, stroma-supporting, keratinized, and vacuolated epithelial cells occur in the thymic medulla. These last two cell types differentiate subsequently into Hassall's bodies and hypertrophied cells. Lympho-epithelial cell complexes are identified in the deep cortex around birth, when the cortical parenchyma houses a transitional erythropoiesis. mAbs (His-39, RMC-20) which recognize medullary epithelial cells in the adult thymus stain positively cells of the thymic primordium as early as day 16 of embryonic life. Cortical epithelial cell markers (His-37, RMC-17) appear, however, slightly later and the subcapsulary region is not established until postnatal life. MHC class I and class II molecules can be identified on epithelial cells in the thymus of 15-day-old embryonic rats although they reach the highest expression around birth. CONCLUSIONS: Our results confirm the heterogeneity of the thymic epithelial component, the persistence of primitive, non-differentiated epithelial cells morphologically similar to those occurring in the early thymic primordium in adult thymus, and the mutual relevance of epithelial cells and thymocytes for an adequate development of rat thymus gland.

Macrophage-lymphocyte cell clusters in the hypothalamic ventricle of some elasmobranch fish: ultrastructural analysis and possible functional significance.

BACKGROUND: Previous studies have demonstrated the existence of lympho-haemopoietic tissue in the meninges and choroid plexuses of various primitive vertebrates, including the stingray Dasyatis akajei and in early human embryos. In the present study, we extend these results analyzing macrophage-lymphocyte cell clusters found in the floor of the hypothalamic ventricle of several specimens of elasmobranchs. METHODS: After aseptical isolation of the brain from several specimens of smooth dogfish Triakis scyllia, cloudy dogfish Scyliorhinus torazame, gummy shark Mustelus manazo, and stingray Dasyatis akajei their hypothalamic regions were processed routinely by light, scanning, and transmission electron microscopy. RESULTS: The study of serial histological sections demonstrated that the macrophage-lymphocyte cell clusters proceeded from the meningeal lymphohaemopoietic tissue, reaching the ventricular lumen along large blood vessels. In this tissue, macrophages, different sized lymphocytes, lymphoblasts, granulocytes, monocytes, and developing and mature plasma cells were closely packed among a meshwork of fibroblastic reticular cell processes. It never invaded the brain parenchyma. A cell layer of glial elements and a continuous basement membrane interposed between the lymphoid tissue and the neural elements although some macrophages had migrated across the ependymal cell layer. In the ventricular lumen very irregular macrophages with long cell processes and containing abundant engulfed material of unknown origin formed big cell clusters with neighboring lymphocytes, lymphoblasts, and plasma cells, similar to those described during the immune response. Moreover, electron lucent cells which resembled the antigen-presenting cells of higher vertebrates established intimate surface cell contacts with the surrounding lymphocytes. In the third ventricle of several specimens of gummy shark, Mustelus manazo, morphologically similar cell clusters appeared but these were not connected to the meningeal lympho-haemopoietic tissue. No intraventricular cell aggregates were found in the stingray brain. CONCLUSIONS: Although we cannot rule out that these macrophage-lymphocyte cell clusters represent a permanent structure in the elasmobranch brain they rather seem to be only established after specific stimulation for preventing the entrance of noxious, foreign materials into the elasmobranch brain parenchyma.

The relevance of cell microenvironments for the appearance of lympho-haemopoietic tissues in primitive vertebrates.

In higher vertebrates, mainly in mammals, a role for the non-lymphoid components of lymphoid organs in governing the maturation and functioning of immune system has been largely demonstrated. In contrast, such a role in the evolution of the vertebrate immune system has only been evidenced indirectly. In the present review we summarize histophysiological results which emphasize the relevance of lympho-haemopoietic stromal elements in the emergence and evolution of vertebrate lymphoid organs. The most primitive vertebrates, the Agnatha, have no true lymphoid organs and, accordingly, their immune responses seem more related to the non-anticipatory defence mechanisms of invertebrates than to the immune responses of vertebrates. So, the appearance and evolution of vertebrate lymphoid organs seems closely related with the emergence of immune capacities. Thymus, spleen and gut-associated lymphoid organs appear early in phylogeny whereas lymph nodes and bone marrow are late phylogenetical adquisitions. However, bone marrowless vertebrates contain numerous organs (i.e., gonads, kidney, brain, etc...), the cell microenvironments of which support lympho-haemopoiesis mimicking the condition of higher vertebrate bone marrow. On the other hand, the lack of germinal centres, another feature of the lymphoid organs of ectothermic vertebrates which impedes the selection of B cells raised after somatic hypermutation, presumably reflects the absence of some of the elements necessary for this organization.

Ontogeny of rat thymic macrophages. Phenotypic characterization and possible relationships between different cell subsets.

In the present study we combined electron microscopy, immunohistology and primary stromal cell cultures to analyse the ontogeny of rat thymic macrophages (M phi) in an attempt to clarify the relationships between the different macrophage cell subsets described in adult rat thymus. Although phagocytic cells were observed in 15-day-old fetal thymus, monoclonal antibodies (mAb) which recognize different adult macrophage types were unable to identify positive cells until the end of embryonic life. However, our in vitro results from primary thymic stromal cell cultures of 16-day-old fetal rats, and the phenotyping of enriched thymic CD2- cell suspensions, demonstrated that monocyte-like cells which strongly expressed major histocompatibility complex (MHC) class II molecules colonized the embryonic thymus early, giving rise later to distinct macrophage subsets. During the process of maturation, macrophage precursors gradually lost their MHC class II expression, acquired other surface markers (CD45, Thy-1, CD25, CD4, etc.) and increased the acid phosphatase activity. In this respect, ED1+ macrophages, which appeared for the first time in the last stages of embryonic life, consisted of a MHC class II molecule-expressing phagocytic cell population, presumably involved in the elimination of non-selected cortical thymocytes, and of non-phagocytic cells which, in the thymic cortex, might differentiate to ED2+ macrophages throughout ED1+ED2lo/med and ED1+ ED2high intermediate cell stages, observed in vitro in 16-day-old fetal thymic stromal cell cultures. At the end of embryonic life and during the postnatal period the numbers of thymic macrophages increased, particularly in the medulla and corticomedullary border (CMZ), and more slowly in the thymic cortex. This increase was presumably due to the arrival, through perivascular spaces, of new macrophage progenitors, rather than in situ proliferation of pre-existent mature macrophages. The possible function of different thymic macrophage subsets, as well as the relationships between themselves and with their presumptive monocyte-like precursors, are discussed.