A conceptual and practical overview of cDNA microarray technology: implications for basic and clinical sciences.

cDNA microarray is an innovative technology that facilitates the analysis of the expression of thousands of genes simultaneously. The utilization of this methodology, which is rapidly evolving, requires a combination of expertise from the biological, mathematical and statistical sciences. In this review, we attempt to provide an overview of the principles of cDNA microarray technology, the practical concerns of the analytical processing of the data obtained, the correlation of this methodology with other data analysis methods such as immunohistochemistry in tissue microarrays, and the cDNA microarray application in distinct areas of the basic and clinical sciences.

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.

Growth hormone and its receptor are expressed in human thymic cells.

GH has been shown to modulate various functions of the thymus. We now demonstrate the production of human GH (hGH) by human thymic cells, and the expression of GH receptors in thymic epithelial cells (TEC) and in thymocytes at different stages of differentiation. The presence of hGH messenger RNA was shown by RT-PCR in both human thymocytes and in primary cultures of TEC. Moreover, immunoreactive hGH material was detected in culture media of thymocytes and TEC with the use of a sensitive immunoradiometric assay. GH receptor gene expression was shown in TEC in primary cultures and in fetal and postnatal TEC lines as well as in thymocytes. By immunocytochemistry, the presence of GH receptors in the various TEC preparations was confirmed. In cytofluorometric studies with the use of a biotinylated anti-GH receptor monoclonal antibody, we could show that GH receptors are predominantly expressed by immature thymocytes: over 90% of CD3- CD4- CD8- CD19- CD34+ CD2- cells (a phenotype characterizing the most immature T cell progenitors in the thymus) were GH receptor positive. Our results provide a molecular basis for an autocrine/paracrine mode of action of GH in the human thymus.

Growth hormone stimulates the proliferation of activated mouse T lymphocytes.

A modulatory role for GH on immune function has been suggested, but hormonal effects have been difficult to demonstrate with isolated cells. We have recently shown that GH receptors are present in murine hematopoietic tissues, with a lower receptor number in T lymphocytes than in B cells or macrophages. The binding of bovine GH (bGH) to murine splenocytes is increased after T cell activation with either concanavalin A or anti-CD3 antibody. In the present study, we show that bGH is able to stimulate the proliferation of activated murine T cells. Splenocytes were stimulated with either Con A or anti-CD3 antibody; addition of the mitogen resulted in increased [3H]thymidine uptake. When added together with the mitogen to the culture medium, bGH was able to further stimulate thymidine uptake. A bell-shaped dose-response curve was observed. bGH was able to increase cell proliferation by 2.5-fold over the effect of anti-CD3 alone. The amplitude of the bGH response was greater in unfractionated splenocytes than in purified T lymphocytes or thymocytes. Splenocytes were also stimulated by lipopolysaccharide, a B cell-specific mitogen; no change in the level of bGH binding was observed during activation of B cells, and no effect of bGH on the proliferative response of splenocytes to lipopolysaccharide was detected. The GH proliferative effect on T lymphocytes is probably direct and not through locally produced insulin-like growth factor I, because insulin-like growth factor I did not affect the cell proliferation when added at concentrations ranging from 10(-9)-10(-7) M. Ovine PRL was also able to stimulate [3H]thymidine uptake in splenocytes and thymocytes, and a synergistic effect was observed when bGH and ovine PRL were added together at 10(-8) M. Our findings support the biological significance of the GH receptors identified in murine T lymphocytes and confirm the role of GH in the regulation of immune functions.

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.

Is there a role for growth hormone upon intrathymic T-cell migration?

Intrathymic T-cell differentiation is essentially driven by the thymic microenvironment, a tridimensional network formed by thymic epithelial cells and to a lesser extent, dendritic cells, macrophages, fibroblasts, and extracellular matrix components. Thymocyte migration throughout the thymus is partially dependent on extracellular-matrix (ECM)-mediated interactions. Herein we investigated the putative role of growth hormone (GH) upon events related to intrathymic T-cell migration. We demonstrated that GH upregulates the expression of ECM ligands and receptors in distinct preparations of cultured thymic epithelial cells TECs). We also showed that adhesion of thymocytes to thymic epithelial cells was significantly increased by GH treatment, an effect that could be consistently abrogated when TECs were treated to antifibronectin, anti-VLA5, antilaminin, or anti-VLA6 antibodies before addition of thymocytes to the cultures. We also studied thymic nurse cells (TNCs), lymphoepithelial complexes that can be isolated ex vivo from the thymus. In this system, we had previously demonstrated that ECM ligands and receptors control both inward and outward thymocyte traffic. We then showed that GH enhances thymocyte release from TNCs, as well as the reconstitution of these lymphoepithelial complexes. Lastly, we evaluated the in vivo influence of GH on thymocyte exit. This was done by means of intrathymic injection of GH plus fluorescein isothiocyanate (FITC), and further analysis of recent thymic emigrants (FITC+ cells) in peripheral lymphoid organs, as defined by CD4/CD8-based cytofluorometric phenotyping. The proportions of FITC+ T cells appeared augmented in lymph nodes in GH-treated mice, as compared to controls. Taken together, these data indicate that GH stimulates intrathymic T-cell traffic, an effect that is at least partially mediated by extracellular matrix-mediated interactions.

Growth hormone receptors and immunocompetent cells.

Growth hormone plays a significant role in regulation of the humoral and cellular immune responses in physiological as well as pathological situations. This role is exerted via the existence of specific receptors on cells of the immune system. Using flow cytofluorometry and biotinylated bovine GH, we have recently analyzed the expression of GH receptors (GHRs) in murine lymphoid organs. GHRs are widely expressed in all murine hematopoietic tissues and in fetuses, newborns, and 3- and 7-week-old animals. In the bone marrow, all hematopoietic lineages express variables levels of GH receptors, whereas in the thymus, this expression is mainly seen in CD4-, CD8-, CD4+CD8+, and CD8+ subpopulations. At the periphery, 50% of splenocytes and peripheral blood lymphocytes and 20% of lymph node cells are GHR positive, with a wider receptor expression on B cells and macrophages (approximately 50%) than on T cells (approximately 20%), where the labeling is seen on both CD4+ and CD8+ cell subsets. Interestingly, the proportion of GHRs bearing CD4+ and CD8+ splenocytes is increased after T-cell activation with Con A or anti-CD3. Finally, all peripheral T cells expressing GHRs also express prolactin receptors. These data provide a molecular basis to study the factors controlling GHR expression and regulation of immune function.

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 which bone marrow-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 secretions 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, primarily 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, which 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 apparently mediated by galectin 3 (an endogenous soluble beta-galactoside-binding lectin).(ABSTRACT TRUNCATED AT 250 WORDS)

Triiodothyronine modulates differential homing of recent thymic emigrants to peripheral lymphoid organs.

The functioning of the immune system partially relies on T-cell exportation from the thymus, the major site of T-cell differentiation. Although the molecular mechanisms governing this process begin to be elucidated, it is not clear if thyroid hormones can alter the homing of recent thymic emigrants (RTE) to peripheral lymphoid organs. Herein, we investigated whether triiodothyronine (T(3)) could influence the homing of thymus-derived T cells. For that we used intrathymic injection of T(3) in combination with fluorescein isothiocyanate (FITC) to trace, 16 h later, FITC(+) cells, termed RTE, in peripheral lymphoid organs. We observed that T(3) stimulated thymocyte export, increasing the frequency of CD4(+) RTE and CD8(+) RTE in the subcutaneous and mesenteric lymph nodes. By contrast, the relative numbers of CD4(+) RTE in the spleen were decreased. T(3) also changed the differential distribution pattern of CD4(+) RTE, and to a lesser extent CD8(+) RTE in the peripheral lymphoid organs. Moreover, the expression of extracellular matrix (ECM) components, such as laminin and fibronectin, which are known to be involved in T-cell migration, increased in the lymph nodes but not in the spleen following intrathymic T(3) treatment. In conclusion, our data correspond to the first demonstration that in vivo treatment with thyroid hormone stimulates thymic T-cell homing and T-cell distribution in peripheral lymphoid organs.

Triiodothyronine modulates thymocyte migration.

Triiodothyronine (T(3)) exerts several effects on thymus physiology. In this sense, T(3) is known to stimulate thymic microenvironmental cells to enhance the production of extracellular matrix (ECM) moieties, which are relevant in thymocyte migration. Here, we further investigated the in vivo influence of T(3) on ECM production, as well as on ECM-related T-cell migration events. For this, BALB/c mice were subjected to two protocols of T(3) treatment: long-term (30 days) i.p. daily T(3) injections or short-term (16 h) after a single T(3) intrathymic injection. These two treatments did promote an enhancement in the expression of fibronectin and laminin, in both cortex and medullary regions of the thymic lobules. As revealed by the long-term treatment, the expression of ECM protein receptors, including VLA-4, VLA-5 and VLA-6, was also increased in thymocyte subsets issued from T(3)-treated mice. We further used thymic nurse cells (TNC) as an in vitro system to study the ECM-related migration of immature thymocytes in the context of thymic epithelial cells. Even a single intrathymic injection of T(3) resulted in an increase in the ex vivo exit of thymocytes from TNC lymphoepithelial complexes. Accordingly, when we evaluated thymocyte migration in transwell chambers pre-coated with ECM proteins, we found an increase in the numbers of migrating cells, when thymocytes were derived from T(3)-treated mice. Overall, our data show that in vivo intrathymic short-term i.p. long-term T(3) treatments are able to modulate thymocyte migration, probably via ECM-mediated interactions.

Control of the thymic microenvironment by growth hormone/insulin-like growth factor-I-mediated circuits.

The thymus gland is a central lymphoid organ in which bone marrow-derived T cell precursors undergo maturation, eventually leading to the migration of positively selected thymocytes to the T-dependent areas of peripheral lymphoid organs. This process occurs under the influence of the thymic microenvironment, by means of secretory polypeptides and cell-cell contacts. The thymic microenvironment is a tridimensional cellular network composed of epithelial cells (its major component), macrophages, dendritic cells, fibroblasts and extracellular matrix elements. The epithelial reticulum is a heterogeneous tissue, in which a particular lymphoepithelial structure has been isolated in vitro: the thymic nurse cell complex, which possibly creates particular microenvironmental conditions for thymocyte differentiation. Additionally, thymic nurse cells are useful tools to study mechanisms involved in intrathymic T cell migration, including neuroendocrine influences. Previous data showed that thymic hormonal function can be modulated by hormones and neuropeptides, including growth hormone. Interestingly, GH acts pleiotropically on the thymic epithelium increasing cell growth and expression of extracellular matrix ligands and receptors, the latter resulting in an enhancement of thymocyte adhesion to the epithelial cells and thymocyte release from thymic nurse cells. The role of GH on thymus development is further stressed by the findings obtained with GH-deficient dwarf mice. Besides the precocious decline in serum thymulin found in these animals, a progressive thymic hypoplasia occurs, with decreased numbers of CD4+CD8+thymocytes, both defects being largely restored by long-term GH treatment. The effects of GH in the thymus are apparently mediated by IGF-1. Enhancement of thymulin secretion induced by GH, as well as the stimulation of thymocyte adhesion to thymic epithelial cells can be prevented in vitro by treatment with antibodies for IGF-I or IGF-I receptor. Moreover, in both systems IGF-I alone can yield similar effects. Also, the enhanced concanavalin-A mitogenic response and IL-6 production by thymocytes observed in GH-treated mice can be detected in animals treated with IGF-I. Lastly, mouse substrains selected for high or low IGF-I circulating levels exhibited differential thymus developmental patterns correlating with IGF-I levels. A further conceptual aspect concerning the GH-IGF-I-mediated control of thymus physiology is the recent demonstration of an intrathymic production of these molecules, leading to the hypothesis that, in addition to the classical endocrine pathway, GH-IGF-I-mediated paracrine and autocrine pathways may also be implicated in the control of thymus physiology. In any case, such control is exerted pleiotropically, with modulation in the expression of several genes in different cell types of the organ. In this respect, it is exciting to imagine a role of GH-IGF-I loops in shaping the intrathymically generated T cell repertoire.

Role of prolactin and growth hormone on thymus physiology.

Intrathymic T-cell differentiation is under the control of the thymic microenvironment, which acts on maturing thymocytes via membrane as well as soluble products. Increasing data show that this process can be modulated by classical hormones, as exemplified herein by prolactin (PRL) and growth hormone (GH), largely secreted by the pituitary gland. Both PRL and GH stimulate the secretion of thymulin, a thymic hormone produced by thymic epithelial cells. Conversely, low levels of circulating thymulin parallel hypopituitary states. Interestingly, the enhancing effects of GH on thymulin seem to be mediated by insulinlike growth factor 1 (IGF-1) since they can be abrogated with anti-IGF-1 or anti-IGF-1-receptor antibodies. The influence of PRL and GH on the thymic epithelium is pleiotropic: PRL enhances in vivo the expression of high-molecular-weight cytokeratins and stimulates in vitro TEC proliferation, an effect that is shared by GH and IGF-1. Differentiating T cells are also targets for the intrathymic action of PRL and GH. In vivo inoculation of a rat pituitary cell line into old rats results in restoration of the thymus, including differentiation of CD4- CD8- thymocytes into CD4+ CD8+ cells. Furthermore, PRL may regulate the maintenance of thymocyte viability during the double-positive stage of thymocyte differentiation. Injections of GH into aging mice increase total thymocyte numbers and the percentage of CD3-bearing cells, as well as the Concanavalin-A mitogenic response and IL-6 production by thymocytes. Interestingly, similar findings are observed in animals treated with IGF-1. Lastly, the thymic hypoplasia observed in dwarf mice can be reversed with GH treatment. In keeping with the data summarized earlier is the detection of receptors for PRL and GH on both thymocytes and thymic epithelial cells. Importantly, recent studies indicate that both cell types can produce PRL and GH intrathymically. Similarly, production of IGF-1 and expression of a corresponding receptor has also been demonstrated. In conclusion, these data strongly indicate that the thymus is physiologically under control of pituitary hormones PRL and GH. In addition to the classical endocrine pathway, paracrine and autocrine circuits are probably implicated in such control.

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.

A conceptual and practical overview of cDNA microarray technology: implications for basic and clinical sciences

cDNA microarray is an innovative technology that facilitates the analysis of the expression of thousands of genes simultaneously. The utilization of this methodology, which is rapidly evolving, requires a combination of expertise from the biological, mathematical and statistical sciences. In this review, we attempt to provide an overview of the principles of cDNA microarray technology, the practical concerns of the analytical processing of the data obtained, the correlation of this methodology with other data analysis methods such as immunohistochemistry in tissue microarrays, and the cDNA microarray application in distinct areas of the basic and clinical sciences.

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.