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Neuroendocrine Control of Thymus Physiology¹

Laboratory on Thymus Research (W.S.), Department of Immunology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, 21045–900 Rio de Janeiro, Brazil; and CNRS UMR 8603 (M.D.), Université Paris V, Hôpital Necker, 75015 Paris, France

	Abstract

Top Abstract I. Introduction II. The Thymic Microenvironment... III. Neuroendocrine Control of... IV. Thymic Endocrine Function... V. Proliferation of Thymic... VI. Hormonal Modulation of... VII. Is Thymocyte Traffic... VIII. Expression of Receptors... IX. Intrathymic Expression of... X. Conclusions and Major... References

 
The thymus gland is a central lymphoid organ in which bone marrow-derived T cell precursors undergo differentiation, eventually leading to migration of positively selected thymocytes to the peripheral lymphoid organs. This differentiation occurs along with cell migration in the context of the thymic microenvironment, formed of epithelial cells, macrophages, dendritic cells, fibroblasts, and extracellular matrix components. Various interactions occurring between microenvironmental cells and differentiating thymocytes are under neuroendocrine control. In this review, we summarize data showing that thymus physiology is pleiotropically influenced by hormones and neuropeptides. These molecules modulate the expression of major histocompatibility complex gene products by microenvironmental cells and the extracellular matrix-mediated interactions, leading to enhanced thymocyte adhesion to thymic epithelial cells. Cytokine production and thymic endocrine function (herein exemplified by thymulin production) are also hormonally controlled, and, interestingly in this latter case, a bidirectional circuitry seems to exist since thymic-derived peptides also modulate hormonal production.

In addition to their role in thymic cell proliferation and apoptosis, hormones and neuropeptides also modulate intrathymic T cell differentiation, influencing the generation of the T cell repertoire.

Finally, neuroendocrine control of the thymus appears extremely complex, with possible influence of biological circuitry involving the intrathymic production of a variety of hormones and neuropeptides and the expression of their respective receptors by thymic cells.

I. Introduction

II. The Thymic Microenvironment and Its Role in T Cell Differentiation

A. Intrathymic T cell differentiation: general comments

B. Cellular interactions involving the thymic microenvironment

C. Heterogeneity of the thymic epithelium: the thymic nurse cell complex

III. Neuroendocrine Control of Membrane Interactions Between Thymocytes and Microenvironmental Cells

A. Is there a hormonal effect upon MHC expression by microenvironmental cells?

B. Extracellular matrix-mediated TEC-thymocyte interactions are hormonally modulated

C. Are inter-TEC gap junctions under neuroendocrine control?

IV. Thymic Endocrine Function and Cytokine Secretion by Microenvironmental Cells Are Controlled by Hormones and Neuropeptides: The Paradigm of Thymulin

A. Steroid and peptidic hormones influence in vitro cytokine secretion

B. Thyroid and pituitary hormone status modulates thymulin secretion

C. Effects of adrenalectomy and gonadectomy on thymulin levels

D. Is there an autocrine/paracrine circuitry controlling thymulin secretion?

E. Thymic hormones modulate endocrine glands and neuroendocrine circuits

V. Proliferation of Thymic Cells Is Hormonally Influenced

A. In vitro effects of pituitary hormones on TEC growth

B. Modulation of thymocyte proliferation by hormones and neuropeptides

VI. Hormonal Modulation of Intrathymic T Cell Differentiation

A. Thymocyte-derived cytokine profile

B. Changes in the T cell differentiation markers and TCR Vß repertoire

C. Hormone-mediated apoptosis in thymocytes

VII. Is Thymocyte Traffic Under Neuroendocrine Control?

A. Effects on the entrance of cell precursors into the thymus

B. Modulation of thymocyte traffic in TNCs

C. Control of thymocyte exit from the thymus

VIII. Expression of Receptors for Hormones and Neuropeptides by Thymic Cells

A. The steroid/thyroid hormone receptor family

B. Expression of PRL and GH receptors by thymocytes and TEC

C. Receptors for neuropeptides in thymic cells

IX. Intrathymic Production of Hormones and Neuropeptides

A. Intrathymic production of corticosterone: role in the shaping of the T cell repertoire

B. Expression of "classic" adenopituitary hormones by thymic cells

C. Is there a functional autocrine/paracrine IGF-I-mediated circuitry in the thymus?

D. Neuropeptide expression by the thymic microenvironment

X. Conclusions and Major Questions to Be Addressed

	I. Introduction

Top Abstract I. Introduction II. The Thymic Microenvironment... III. Neuroendocrine Control of... IV. Thymic Endocrine Function... V. Proliferation of Thymic... VI. Hormonal Modulation of... VII. Is Thymocyte Traffic... VIII. Expression of Receptors... IX. Intrathymic Expression of... X. Conclusions and Major... References

 
THE CROSS-TALK between the neuroendocrine and immune systems is now well demonstrated. These systems use similar ligands and receptors to establish a physiological intra- and intersystem communication circuitry that plays an important role in homeostasis. Increasing evidence has placed hormones and neuropeptides among potent immunomodulators, participating in various aspects of immune system function, in both health and disease (reviewed in Refs. 1, 2, 3, 4). More particularly, the physiology of the thymus is modulated by a variety of biological circuits including those mediated by steroid and polypeptidic hormones, as well as neuropeptides. Herein, we focus on both the microenvironmental and the lymphoid aspects of neuroendocrine control of the thymus. We will first briefly comment on the structure of the thymic microenvironment and its role in the complex process of intrathymic T cell differentiation.

	II. The Thymic Microenvironment and Its Role in T Cell Differentiation

Top Abstract I. Introduction II. The Thymic Microenvironment... III. Neuroendocrine Control of... IV. Thymic Endocrine Function... V. Proliferation of Thymic... VI. Hormonal Modulation of... VII. Is Thymocyte Traffic... VIII. Expression of Receptors... IX. Intrathymic Expression of... X. Conclusions and Major... References

 
The thymus gland is a central lymphoid organ in which bone marrow-derived T cell precursors undergo a complex process of maturation, eventually leading to migration of positively selected thymocytes to the T cell-dependent areas of peripheral lymphoid organs such as spleen, lymph nodes, Peyer’s patches, and tonsils (5). Such a differentiation process involves sequential expression of various membrane markers and rearrangements of the T cell receptor genes.

Importantly, although thymocyte proliferation and differentiation persist throughout life, they diminish with aging. Older thymuses are significantly atrophied and have fewer thymocytes than younger ones. However, even in humans, the adult thymus is still active in terms of delivering mature T lymphocytes to the periphery of the immune system (6, 7).

A. Intrathymic T cell differentiation: general comments
From the entrance of T cell precursors into the thymus to the exit of mature cells from the organ, a vast body of interactions promotes the complex process of T cell differentiation. This differentiation involves regulation of the expression of various membrane proteins. A key membrane protein is the T cell receptor (TCR), which in the cell membrane is physiologically coupled with a molecular complex, termed CD3. Additional accessory molecules, including CD4 and CD8, as well as CD25 and the proteoglycan CD44, are useful to define stages of intrathymic T cell differentiation.

The TCR is a heterodimer formed by an ß- or a -chain configuration. Although ⁺ thymocytes are the first to appear in the thymus with ontogeny of the organ, in the adult organ, around 99% of TCR⁺ thymocytes express TCRß and only 1% are T cells. A major point in intrathymic T cell differentiation is that, after gene rearrangements, any of the TCR peptide chains are generated, resulting in a large diversity of TCRs bearing distinct peptide specificities. This complex but well understood phenomenon is beyond the scope of this article but is reviewed in detail in recent publications (8, 9, 10).

The CD3 complex is an assembly of polypeptidic chains physically associated with the TCR. This association, together with the fact that CD3 bears cytoplasmic domains capable of phosphorylation, provides the intracellular signal transduction pathways necessary for TCR-driven T cell activation. Such activation follows ligation with a peptide presented by molecules of the major histocompatibility complex (MHC) expressed on the membranes of nonlymphoid cells. This ligation is favored by the accessory molecules CD4 and CD8, which are transmembrane glycoproteins that interact with class II and class I MHC molecules, respectively.

CD25 is the -chain of the interleukin 2 (IL-2) receptor, and when it is expressed together with the ß- and -chain, the receptor acquires high affinity for IL-2, thus favoring IL-2-driven thymocyte proliferation. The proteoglycan CD44 is a receptor for hyaluronic acid, and to a lesser extent for fibronectin and collagen. As seen below, it is associated with thymocyte migration events and is also considered a marker for T cell activation (11).

Cytofluorometric combined analysis of these markers proved to be useful in defining intrathymic T cell differentiation. The most immature thymocytes express neither the TCR/CD3 complex nor the accessory molecules CD4 or CD8 and thus are called double-negative thymocytes. Nevertheless, we can determine differentiation steps within the double-negative compartment by distinguishing the cells on the basis of their CD25 and CD44 expression. Thymocyte precursors that recently entered the thymus, in addition to being TCR/CD3^-CD4^-CD8^-, are also CD44⁺CD25^-. As they differentiate, these immature cells acquire CD25 on the cell membrane, becoming CD44⁺CD25⁺, and then sequentially lose CD44 and CD25. The whole double-negative compartment represents about 5% of total thymocytes. Thymocyte maturation then progresses with the acquisition of both CD4 and CD8 markers, generating the so-called CD4⁺CD8⁺ double-positive thymocytes. These cells are the most common in the thymus, comprising 80% of total thymocytes. In the double-positive stage, TCR genes are rearranged. In differentiation of TCRß-bearing cells, the ß-chain-related genes are rearranged first followed by the -chain genes. At this stage, TCR is expressed in low density on the cell membrane. Thymocytes that do not undergo a productive TCR gene rearrangement (i.e., that will not ultimately generate a peptide chain expressed on the cell membrane) die by default through apoptosis. By contrast, those expressing productive TCR will be able to react with peptides presented by molecules of the MHC, expressed on the membranes of nonlymphoid cells. This interaction will determine the positive and negative selection events, crucial for normal thymocyte differentiation. Positive selection allows the differentiation step whereby an immature, short-lived, CD4⁺CD8⁺ thymocyte escapes from programmed cell death and becomes a mature, long-lived, CD4⁺ or CD8⁺ single positive cell. This is a highly stringent process, sparing only a small proportion of the CD4⁺CD8⁺ population. Positive selection also coincides with lineage commitment: the decision to become a CD4⁺ or CD8⁺ single positive thymocyte, as a function of the class of MHC molecule with which the TCR can interact. Negative selection in the thymus is the screen for establishing self-tolerance in the T cell repertoire, promoting deletion of T cells that might potentially be autoreactive to self-proteins. As illustrated in Fig. 1, positive selection events begin earlier in CD3⁺ double-positive cells, whereas negative selection takes place in both double-positive and single-positive thymocytes (Reviewed in Ref. 8).

View larger version (93K): [in this window] [in a new window]   Figure 1. Stages of thymocyte differentiation. This scheme depicts the differentiation pathway for TCRß+ mouse thymocytes, as ascertained by the expression of various surface markers. When entering the thymus, T cell precursors bear the phenotype TCR/CD3-CD4-CD8-CD25-CD44+. As they differentiate, immature cells acquire CD25, become CD44+CD25+, and then gradually lose CD44 followed by loss of CD25. Thymocyte maturation then progresses with the acquisition of CD4 and CD8 markers, generating the CD4+CD8+ thymocytes. In the double-positive stage, TCR genes are rearranged, for sequential expression of ß- and -chains, respectively (illustrated by the intra-arrow wheels in the figure). Productive TCRs, complexed with CD3, are initially expressed in low density on the cell membranes. These cells are then exposed to positive and negative selection events. Positively selected thymocytes progress to the mature CD4+CD8- or CD4-CD8+ single positive stage. These mature thymocytes, which now express high densities of the TCR/CD3 complex, will ultimately leave the organ. Thymocytes that did not succeed in expressing TCR on the cell membrane, as well as those undergoing negative selection, will die by apoptosis. Localization of these various steps of thymocyte differentiation in the thymic lobule can be seen at the right side of the figure.

It is noteworthy that thymocyte differentiation occurs as cells migrate within the thymic lobules. As seen in Fig. 2, top panel, most of the immature thymocytes, including those bearing the phenotypes CD3^-CD4^-CD8^- and CD3⁺CD4⁺CD8⁺ are cortically located, whereas mature CD3⁺CD4⁺CD8^- and CD3⁺CD4^-CD8⁺ cells are found in the medulla, being those that will normally leave the organ to populate the T cell-dependent areas of peripheral lymphoid organs (5).

View larger version (87K): [in this window] [in a new window]   Figure 2. The thymic microenvironment and intrathymic T cell differentiation. A simplified model of thymocyte migration can be seen in the top panel. This panel depicts the common entrance site of precursor cells into the organ through blood vessels. Having entered the thymus, thymocytes migrate during differentiation to ultimately leave the organ (also by blood vessels), bearing the mature phenotypes of CD4+CD8- or CD4-CD8+ thymocytes. The bottom panel schematically depicts a thymic lobule, showing thymocytes intermingled with a heterogeneous cellular network representing the thymic microenvironment. This nonlymphoid compartment is composed of various cell types, including epithelial cells (yellow and orange), dendritic cells (red), macrophages (blue), and fibroblasts (green). Among TEC, morphological heterogeneity can be distinguished in subseptal/subcapsullary, cortical, and medullary regions. A particular cortically located lymphoepithelial complex, the thymic nurse cell, is seen.

B. Cellular interactions involving the thymic microenvironment
Several kinds of heterotypic interactions occur between differentiating thymocytes and microenvironmental cells. As mentioned above, one key cellular interaction involves the TCR/CD3 complex, expressed by differentiating thymocytes, with class I or class II MHC products on the microenvironmental cell membranes, complexed with a given endogenous peptide to be recognized, in the context of CD8 or CD4 molecules, respectively. The avidity of the resulting interaction is a determinant for positive vs. negative selection. Thymocytes with high avidity are negatively selected and are also deleted by apoptosis. This leads to the death of large numbers of potentially harmful autoreactive T cells. By contrast, a small percentage of thymocytes with intermediate avidity for recognition of MHC self-peptides appears to be rescued from death and is positively selected. Positive selection appears to be essentially conveyed by thymic epithelial cells (TEC) whereas negative selection can occur in the context of hematopoietic-derived DC, but also of TEC (5, 13).

In addition to the TCR/MHC-peptide interaction, the thymic microenvironment can influence the process of thymocyte migration/differentiation via other types of heterotypic membrane interactions. For example, TEC express classical membrane adhesion molecules such as ICAM-1 (intercellular adhesion molecule 1) and LFA-3, which respectively bind to LFA-1 and CD2 present on thymocytes (14, 15, 16). Moreover, TEC-thymocyte interactions can be mediated by ECM ligands such as fibronectin and laminin and their corresponding integrin receptors VLA-4/VLA-5 and VLA-6 (17, 18, 19). In fact, it is possible that ECM provides a complex macromolecular substrate onto which thymocytes migrate within the organ, following an ordered pattern as if on a conveyor belt (20). More recently, biochemical and functional evidence was provided that TEC communicate with each other by gap junctions, which are formed by proteins of the connexin family and allow direct passage of low molecular weight substances between adjacent cells (21). Microinjection of low molecular weight fluorochromes also revealed functional gap junction-mediated TEC-thymocyte interactions. Based on further findings that have appeared in the literature, we recently postulated that gap junctions may correspond to a novel route for cell-cell communication in the immune system (22).

Thymic microenvironmental cells can influence thymocyte differentiation and proliferation by means of soluble polypeptides. Both TEC and DC produce the cytokine IL-1, which stimulates thymocyte proliferation (23). Actually, various cytokines can be produced by thymic epithelium, including IL-3, IL-6, IL-7, IL-8, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, transforming growth factor-, transforming growth factor-ß (TGF-ß), leukemia inhibitory factor, and stem cell factor (24, 25).

IL-7, in particular, has been proven to be crucial for thymocyte differentiation. For example, it was shown to promote rearrangement of the TCR genes by enhancing the production and activity of recombinases (26). In conjunction, IL-7^-/- as well as IL-7 receptor-deficient mice display a severe reduction in lymphoid development, whereas the transgene incorporation of IL-7 in nude mice induces T cell development (27). In addition to IL-7, SCF (also termed c-Kit ligand) is necessary for early thymocyte differentiation. When fetal thymuses of spontaneously SCF-deficient mice are grafted into normal wild-type recipients, the number of CD3^-CD4^-CD8^- is more than 10 times lower than in wild-type grafts (27).

In addition to classical cytokines, chemokines are also thymic microenvironment-derived secretory products important in thymus physiology. Chemokines correspond to a family of small polypeptidic molecules that control directional migration of leukocytes (reviewed in Ref. 28). Among others, one chemokine, the stromal cell-derived factor (SDF) is highly expressed in the thymus, being produced by stromal cells, particularly in the subcapsular region (29). In keeping with this topography, SDF preferentially attracts immature CD4^-CD8^- and CD4⁺CD8⁺ thymocytes. Conversely, another chemokine, MIP3ß, exerts chemoattraction for mature single positive thymocytes (30). This is in keeping with the differential expression of corresponding chemokine receptors in distinct CD4/CD8-defined stages of thymocyte differentiation.

This leads to the notion that several paracrine circuits involving TEC-derived factors are likely to have differentiating thymocytes as targets. In addition to producing cytokines, TEC secrete chemically defined thymic hormones, including thymosin-1, thymopoietin, and thymulin (31, 32, 33), that can also act upon the general process of thymocyte maturation (reviewed in Refs. 34, 35, 36). For instance, thymulin, a nonapeptide whose biological activity depends on its coupling to zinc (37, 38), is able to enhance thymocyte proliferation and to induce several T cell markers and functions (reviewed in Refs. 34, 35, 36, 39). The circulating levels of thymulin achieve maximal values early in postnatal life and decline with age (40). More recently, it has been shown that thymulin secretion follows a circadian rhythm, peaking during the night (41). The general characteristics of thymulin and its effects on the immune system are summarized in Table 1. For recent reviews on the various thymic hormones, see Refs. 34, 35, 36 .

View larger version (36K): [in this window] [in a new window]   Figure 3. Heterogeneity of interactions between TEC and thymocytes. Left panel shows types of TEC-thymocyte interactions. Among membrane interactions are those involving the MHC complex (in this case MHC class II molecules) expressed by TEC, binding endogenous peptide presented to the TCR/CD3 complex on thymocytes, in the context of accessory molecules (exemplified herein by the CD4, labeled in blue). Additional cell-cell interactions, comprising receptors and coreceptors, are represented by adhesion molecules, namely LFA-3/CD2 and ICAM-1/LFA-1. Moreover, TEC can interact with thymocytes via ECM ligands and receptors (ECM-R), or gap junctions. In addition to membrane interactions, bidirectional TEC-thymocyte exchange of biological signals can be conveyed by secretory products such as cytokines and thymic hormones. Right panel illustrates that homotypic TEC-TEC interactions can also take place, involving adjacent cell membranes as well as soluble molecules.

Macrophages and DCs are hematopoietic-derived cell types and quantitatively are a minor component of the thymic microenvironment. Dendritic cells are preferentially located in the medulla and at the corticomedullary junction of the thymic lobules, whereas macrophages are distributed throughout the lobule (5); they both express MHC class II molecules and can interact with differentiating thymocytes via membrane proteins and cytokines, including IL-1.

Concerning the constitution of the intrathymically generated T cell repertoire, there is strong evidence that dendritic cells of the thymic microenvironment are involved in determining negative selection of the T cell repertoire by thymocyte deletion (13). In contrast, a role for macrophages in negative selection remains to be conclusively demonstrated.

C. Heterogeneity of the thymic epithelium: the thymic nurse cell complex
The thymic epithelial network is a rather heterogeneous tissue in terms of morphology and phenotype, and cells in different locations within the thymic lobules may be responsible for influencing specific steps in T cell maturation (46). One cortically located lymphoepithelial complex, the thymic nurse cell (TNC), has been isolated in vitro. TNCs are lymphoepithelial multicellular structures formed by one TEC, which in mice can harbor 20–200 thymocytes (47), and are located in the cortical region of thymic lobules (48, 49). Most intra-TNC lymphocytes bear the CD4⁺CD8⁺ double-positive phenotype (50), although immature double-negative as well as mature single-positive cells can also be found. Interestingly, TNCs may create special microenvironmental conditions for thymocyte differentiation and/or proliferation, and within this complex distinct interactions apparently occur, including those mediated by soluble products, gap junctions, ECM, and MHC/TCR (reviewed in Ref. 51). Self-antigens appear to be presented to thymocytes within TNC (52), and intra-TNC lymphocyte apoptosis has recently been reported (53, 54). Once settled in culture, TNCs spontaneously release thymocytes, and TNC-derived epithelial cells can reconstitute lymphoepithelial complexes after being cocultured with fetal thymocytes (55). Thus, TNCs constitute an in vitro model of thymocyte migration within the TEC context (18, 19, 45, 51, 56).

Other experimental models have been used to dissect the sequence of acquisition/loss of differentiation markers, as well as their respective roles in intrathymic T cell differentiation. A significant contribution was the generation of genetically engineered mice (57). The in vitro model of fetal thymus organ cultures (FTOC) is also used to study intrathymic T cell differentiation. By day 14 of gestation, only immature CD4^-CD8^- thymocytes are seen, whereas after a 14-day culture of the thymic lobes, differentiation has progressed with the generation of CD4⁺ or CD8⁺ single-positive mature cells (58).

As detailed below, the various intrathymic cellular interactions as well as the in vivo and in vitro experimental models summarized above can be regarded as potential targets for control by hormones and neuropeptides.

	III. Neuroendocrine Control of Membrane Interactions Between Thymocytes and Microenvironmental Cells

Top Abstract I. Introduction II. The Thymic Microenvironment... III. Neuroendocrine Control of... IV. Thymic Endocrine Function... V. Proliferation of Thymic... VI. Hormonal Modulation of... VII. Is Thymocyte Traffic... VIII. Expression of Receptors... IX. Intrathymic Expression of... X. Conclusions and Major... References

 
A. Is there a hormonal effect upon MHC expression by microenvironmental cells?
Few data are so far available to determine whether hormones or neuropeptides alter the expression of MHC gene products by thymic microenvironmental cells. Recent findings suggest that this may be the case. When human peripheral blood leukocytes were subjected to a mixed lymphocyte culture, the levels of cell proliferation and cytotoxic activity were significantly higher in the presence of recombinant GH, respectively 4- and 7-fold compared with controls (59). This is in keeping with previous evidence that GH treatment in children after renal transplantation worsened allograft function (60). In addition, a rise in IFN- secretion and in the number of cells expressing the corresponding mRNA was observed in vitro (6- to 10-fold more cells in GH-treated patients than in controls). In the same study, it was demonstrated that such effects require the presence of autologous antigen-presenting cells. Given the enhancing effect of GH upon IFN- production, together with the cytokine-induced up-regulation of MHC class I and class II expression (42, 43, 44) and the role played by adhesion molecules of thymic microenvironmental cells in MHC-TCR interactions (45, 55), we suggest that the MHC-mediated influence on thymocyte differentiation may be modulated by fluctuations in GH. This suggestion remains a working hypothesis and should be tested in specifically designed experiments.

More direct evidence, recently reported by Sacedon and co-workers (61, 62, 63), showed an effect of glucocorticoid hormones. Thymic dendritic cells treated in vitro with dexamethasone exhibited a slight, yet consistent, increase in the membrane expression of MHC class I molecules. This seems to be specific to class I, since no effect was observed on the expression of MHC class II gene products.

The same research group showed earlier expression of MHC class I and II molecules during thymus ontogeny in rat fetuses whose mothers had been previously adrenalectomized (52). It should be noted that before full development of the hypothalamus-pituitary-adrenal axis, circulating glucocorticoids in the fetus are strictly of maternal origin. Thus, what these experiments tell us is that absence of circulating glucocorticoids in early fetal life accelerates intrathymic MHC expression. Although in this particular work double labeling for simultaneous detection of MHC and cytokeratin was not reported, the micrographs, showing MHC labeling in the whole microenvironmental network, led us to think that the thymic epithelial network may include MHC expression. This idea is supported by the authors’ finding of earlier detection of other known markers for TEC differentiation in fetal thymuses derived from adrenalectomized mothers.

Thus, the findings discussed above argue in favor of hormonal regulation of MHC expression by the thymic microenvironment. Yet, formal demonstration of a hormonal regulation of MHC expression by TEC is still lacking. Nor has the possible influence of hormones and neuropeptides on intrathymic MHC expression been approached in terms of its consequences on MHC-TCR interactions.

B. Extracellular matrix-mediated TEC-thymocyte interactions are hormonally modulated
Initial studies revealed that the intrathymic production of ECM proteins, including fibronectin, laminin, and type IV collagen, was enhanced in vivo in mice injected with hydrocortisone; thickened ECM-containing fibrils were observed in both cortical and medullary regions of the thymic lobules as early as 24 h posttreatment. In the protocol of a single dose injection, this effect was transient, being progressively reversed in parallel with thymocyte expansion. Additionally, augmented amounts of such ECM components were detected in mouse TEC cultures treated with glucocorticoid hormone (64), indicating that the effect of hydrocortisone enhancing ECM in the thymus represents direct activity on the thymic epithelium. Similar results were obtained with sex steroids (64). At variance with these data, however, it was reported that the levels of fibronectin [measured by enzyme-linked immunosorbent assay (ELISA)] in human TEC culture supernatants were not altered after hydrocortisone treatment (65). Considering that this steroid hormone was also shown to enhance ECM receptor on the TEC membranes (18), it is possible that the lack of modulation in the supernatant derives from the augmented levels of the ECM bound on the TEC surface stimulated by hydrocortisone, which would then mask the levels in the culture supernatants.

In a further study, long-term treatment (30 days) with T₃ in mice also yielded changes in the intrathymic distribution profile of ECM proteins, with an increase in thin ECM fibrils (thus differing from the thick fiber pattern seen after glucocorticoid injection), particularly in the cortical region of thymic lobules. Again, such an effect seems to be direct upon TEC, since enhanced ECM production was also seen when T₃ was added to cultures of a murine TEC line and to TNC-derived TEC preparations (66). More recently, similar results were obtained in vitro when various TEC cultures were subjected to PRL, GH, or insulin-like growth factor I (IGF-I) (56). It should be pointed out that, regarding the in vitro models mentioned above, not only were the amounts of fibronectin and laminin enhanced by various hormone treatments, but also was the expression of their corresponding receptors, VLA-5 and VLA-6 (56, 66).

Since thymocyte-TEC adhesion is at least partially mediated by ECM ligands and receptors, we also tested the various hormones cited above, corticosteroid, thyroid, and pituitary hormones, for their ability to modulate such heterotypic cellular interaction. All enhanced the degree of thymocyte adhesion to cultured TEC. Furthermore, regarding pituitary hormones (56), the hormone-induced enhancement of TEC-thymocyte adhesion was abrogated by monoclonal antibodies specific for each hormone or its corresponding receptor, and also by various anti-ECM or anti-ECM receptor antibodies (Fig. 4).

View larger version (36K): [in this window] [in a new window]   Figure 4. ECM-mediated, GH-induced enhancement of thymocyte adhesion to TEC. Freshly isolated mouse thymocytes are left to adhere onto cultures of a mouse TEC line in the absence or presence of GH. Additionally, some GH-treated cultures are incubated with antifibronectin (-FN), anti-VLA5 (-VLA5), antilaminin (-LN), or anti-VLA6 (-VLA-6) monoclonal antibodies before GH treatment. The GH-induced enhancement of thymocyte-TEC adhesion is clearly abrogated by anti-ECM or anti-ECM receptor antibodies. **, P < 0.01. [Adapted with permission from V. de Mello-Coelho et al.: J Neuroimmunol 76:39–49, 1997 (56 ) © Elsevier Science.]

C. Are inter-TEC gap junctions under neuroendocrine control?
Very recent data indicate that gap junctions mediating communication between adjacent TEC can also be under hormonal control. Decreased cell coupling between adjacent TEC (ascertained by diffusion of intracellularly injected low molecular weight fluorochrome) was seen when cultures were treated by the sex steroids testosterone, progesterone, and estrogen, as well as by the pituitary hormones ACTH and GH, and the neuropeptide calcitonin gene-related peptide (CGRP) and substance Y (67, 68). Conversely, results from our laboratory indicate that vasointestinal peptide (VIP), a neuropeptide that increases intracellular cAMP, enhances inter-TEC cell coupling (69). Similar enhancement was seen when cultures were treated with glucocorticoid hormones, an effect that was significantly abrogated by the use of the glucocorticoid receptor antagonist RU486 (70). Nevertheless, a systematic survey to determine which hormones and neuropeptides modulate gap junction opening between adjacent TEC and between TEC and thymocytes has not yet been made. Nor are data so far available concerning putative neuroendocrine control of connexin expression by TEC.

	IV. Thymic Endocrine Function and Cytokine Secretion by Microenvironmental Cells Are Controlled by Hormones and Neuropeptides: The Paradigm of Thymulin

Top Abstract I. Introduction II. The Thymic Microenvironment... III. Neuroendocrine Control of... IV. Thymic Endocrine Function... V. Proliferation of Thymic... VI. Hormonal Modulation of... VII. Is Thymocyte Traffic... VIII. Expression of Receptors... IX. Intrathymic Expression of... X. Conclusions and Major... References

 
A. Steroid and peptidic hormones influence in vitro cytokine secretion
In addition to acting upon membrane interactions, hormones and neuropeptides can modulate the production of cytokines and hormones by thymic microenvironmental cells. Exogenous IL-1ß enhances IL-6 and IL-8 production by cultured human TEC and induces granulocyte macrophage colony stimulating factor (GM-CSF) as well as leukemia inhibitory factor (LIF) production by these cells. Interestingly, hydrocortisone is able to selectively block the latter effect on GM-CSF and LIF, as ascertained by ELISA assessment of cytokines in TEC culture supernatants (65). In a mouse TEC line, retinoic acid promoted enhanced expression of IL-1, IL-6, and IL-7 (71). The production of IL-1 and IL-1ß by bovine nonepithelial thymic microenvironmental cells in vitro was increased by exogenous GH and by PRL (72). It was further shown that secretion of IL-6 is also up-regulated by GH or by PRL treatment, an effect that could be abrogated by the use of the IL-1 receptor antagonist. This indicates that hormonal effects on microenvironmental cell-derived IL-6 secretion are at least partially exerted through the IL-1 production pathway. Autocrine/paracrine control of cytokine production by the thymic microenvironment appears to involve TEC-derived neuropeptides as well. Martens and colleagues (73) demonstrated that the constitutive production of both IL-6 and LIF (but not IL-1ß) by primary cultures of human TEC was enhanced when monoclonal antibodies to oxytocin were added to the cultures. This strongly suggests that the secretion of these TEC-derived cytokines is partially under the control of oxytocin.

Together, the findings discussed above strongly favor the notion that cytokine production by the thymic microenvironment is under neuroendocrine control. Nonetheless, such a notion should be cautiously viewed since corresponding in vivo data are still lacking. In this respect, evaluation of cytokine production by the microenvironmental cells in mice genetically engineered for hyperproduction or lack of hormones or neuropeptides will be certainly worthwhile. The same is true for more precise analysis of IL-7 and of SCF production by thymic microenvironmental cells under various hormonal fluctuations.

B. Thyroid and pituitary hormone status modulates thymulin secretion
The concept of neuroendocrine control of thymic secretory substances has been particularly developed with regard to the production of the thymic hormone thymulin. In a first series of studies we demonstrated that in vivo treatment with T₃ enhanced thymulin secretion by mouse TEC, and that an opposite effect was seen if the animals were treated with propiothiouracil, an inhibitor of thyroid hormone synthesis (74). These results were confirmed by others (75). In aging mice, injection of T₄ increased thymulin serum levels to values found in young individuals (75). In humans, it was shown that patients with hyperthyroidism exhibit higher levels of circulating thymulin, whereas in hypothyroidism the opposite was observed. In both situations, adequate therapy brought thymulin serum levels within normal range (76).

Experiments using TEC cultures showed that the stimulatory effect of thyroid hormones upon thymulin secretion was due to a direct action of the hormone on the epithelial cells (66) and depended on de novo synthesis of thymulin, since it could be abrogated by cycloheximide (77).

Pituitary hormones were also shown to be potent up-regulators of thymulin secretion. For instance, experimental hyperprolactinemia induced by repeated PRL injections increased thymulin levels in both young and old animals. Conversely, administration of bromocriptine, an agonist of the dopamine receptor, which inhibits PRL biosynthesis, promoted a consistent, dose-dependent decrease in thymulin production (78). These results, obtained in mice, are in keeping with data derived from hyperprolactinemic patients bearing pituitary adenomas, who present abnormally high thymulin serum levels (79).

Fluctuations in GH levels also modulate thymulin secretion. Initial studies revealed that dwarf mice exhibit a precocious decay in thymulin levels (80). This is in keeping with more recent data showing that hypophysectomy in rats yielded a profound, although transient, decrease in thymulin serum levels (81). Similarly, low thymulin levels accompanied deficient GH production in children, whereas GH treatment consistently restored this thymic endocrine function, as early as 24 h after injection (82, 83). Moreover, GH treatment induced an increase in thymulin serum levels that correlated with the amounts of circulating IGF-I. In acromegalic patients, high thymulin serum titers also correlated with high IGF-I serum levels (84).

It is noteworthy that the enhancing effects of PRL and GH on thymulin secretion were directly obtained by treating murine or human TEC cultures and were abrogated by corresponding antihormone antibodies (78, 84). In vitro GH effects were abrogated by anti-IGF-I or anti-IGF-I receptor antibodies, thus incriminating TEC-derived IGF-I as a mediator of the GH effects upon TEC (84). This was further supported by findings that both anti-IGF-I and anti-IGF-I receptor antibodies were also able to block GH-dependent enhancement of TEC-thymocyte adhesion (56).

In addition to the effects observed with classical peptidic hormones, we found that endogenous opioids, namely ß-endorphin and Leu-enkephalin, can up-regulate thymulin secretion by cultured TEC (85). In vivo experiments to confirm this notion are still lacking, however. Nor are data available to define whether other neuropeptides influence thymulin secretion.

C. Effects of adrenalectomy and gonadectomy on thymulin levels
The effects of adrenal and gonadal steroids on thymulin secretion appear to be rather more complex. One of the experimental strategies to approach this issue is ablation of the adrenals and/or gonads. When a single surgery (adrenalectomy or gonadectomy) was performed, we observed in both male and female mice a transient fall in thymulin serum levels that lasted 1 month, peaking 1 week postsurgery, and then progressively returning to normal. More impressive, adrenalectomy + castration resulted in a long-term decrease in the levels of circulating thymulin that persisted until 3 months postsurgery, being followed by gradual restoration to normal levels by 6 months after surgery (86). Interestingly, in both single and double surgical procedures, an increase in the intrathymic content of thymulin was seen. Concomitant to the sustained low levels of the thymic hormone in these experimental conditions, an endogenous low molecular weight thymulin inhibitor was transiently detected in the mouse sera, with concentrations peaking when thymulin levels were lowest. Although the biochemical nature of such a thymulin inhibitor was not defined, its appearance was thymus dependent, since it was not found in mice undergoing thymectomy before adrenalectomy + gonadectomy (86). This series of experiments, although not conclusive, pointed to a rather complex mechanism involved in in vivo steroid hormone influence upon thymic hormone production, possibly comprising other biological circuits, including the hypothalamus-pituitary axis.

This possibility led us to study the influence of steroid hormones on murine and human TEC cultures, and we observed that physiological concentrations of glucocorticoid hormones, estradiol, progesterone, or testosterone, enhanced thymulin release into the culture supernatants. This effect was abrogated when TEC were simultaneously incubated with a given steroid hormone plus the specific antagonist of the corresponding hormone receptor (87).

In view of these findings, it is possible that the transient in vivo increase in the intrathymic contents of thymulin observed after adrenalectomy and/or gonadectomy corresponds to a TEC response to the fall in circulating levels of biologically active thymulin, secondary to the appearance of its natural inhibitor. Such a feedback circuit with increase in thymulin production had been previously shown in mice treated with antithymulin monoclonal antibodies (88).

D. Is there an autocrine/paracrine circuitry controlling thymulin secretion?
That in vivo treatment of mice with antithymulin monoclonal antibodies resulted in an increase in the intrathymic content of the hormone suggested that the level of circulating thymulin could influence its rate of secretion. Similarly, in keeping with these findings, we noted that incubation of cultured TEC with antithymulin monoclonal antibodies resulted in increased numbers of thymulin-containing cells (89). Conversely, thymulin release into TEC culture supernatants can be down-regulated by exogenous addition of the hormone itself.

Interestingly, IL-1, which is also produced in vivo and in vitro by TEC, is able to stimulate in vivo zinc uptake by the thymus (likely to be due to the increase in metallothionein biosynthesis by the epithelial cells), thus up-regulating thymulin secretion in vitro (90, 91).

Altogether, the data discussed above clearly indicate that the general control of thymulin secretion may be very complex, involving distinct biological circuits whose overall balance will dictate the amounts of thymulin to be secreted at a given moment. Due to this apparent complexity, it is predictable that compensatory loops may be triggered when one or more thymulin-controlling axes are disturbed. This would explain why in some experimental situations, fluctuations in thymulin serum levels are transient.

E. Thymic hormones modulate endocrine glands and neuroendocrine circuits
The concept of bidirectionality between the neuroendocrine and immune systems can also be applied to analysis of thymic hormones, since these substances modulate the production of hormones and neuropeptides of the hypothalamus-pituitary axis and some of their target endocrine glands.

Initial experiments revealed that neonatal thymectomy promotes developmental atrophy of female sexual organs (92). One might argue that such an effect could reflect an autoimmune process rather than a direct action of thymic hormones on the neuroendocrine system. This assumption is based on the fact that perinatal thymectomy in BALB/c mice induces autoimmune disease (93). Nevertheless, in the experiments supporting this view, thymectomy was performed on day 3 postnatally, when the thymus has already released a significant amount of thymocytes (initiated on the day of birth). By contrast, in the former experiments, thymectomy was carried out at birth, thus before colonization of peripheral lymphoid organs by T cells. Moreover, as detailed below, it has been shown that production of sex steroids is enhanced in vivo and in vitro by a single thymic hormone, thymulin.

In addition to the action on sexual organs, thymectomy at birth promoted a decrease in the number of secretory granules in acidophilic cells of the adenopituitary (94). This is in keeping with data showing that athymic nude mice exhibit significantly low levels of various pituitary hormones, including PRL, GH, LH, and FSH (95).

Regarding the effects of thymic peptides, it was shown that thymosin-ß4, when perfused intraventricularly, stimulates in vivo LH and its hypothalamic-releasing hormone LHRH (96). A similar stimulation of LH release was obtained with thymulin in perifused or fragmented pituitary preparations (97, 98). Another thymosin component, the MB-35 peptide, also enhanced PRL and GH production (99). In vivo studies in children showed that administration of thymopoietin increases GH and cortisol serum levels (100). Moreover, thymopentin (the synthetic biologically active peptide of thymopoietin) enhanced in vitro the production of POMC derivatives such as ACTH, ß-endorphin, and ß-lipotropin (101). Thymulin exhibited a similar in vitro stimulatory effect on perifused rat pituitaries, enhancing the release of GH, PRL, and, to a lesser extent, TSH (102). With regard to its effect on GH release, it has been shown to be age-dependent, being less efficient in pituitary cell cultures derived from senescent animals (103). The same study showed that this effect of thymulin is mediated by calcium influx, as well as cAMP and inositol phosphate (103). However, contrasting results were reported using short-term cultures of pituitary fragments: a consistent increase in ACTH secretion after in vitro thymulin treatment, with no changes in GH levels but a significant inhibition of PRL release (98).

Thymosin-1 was apparently able to down-regulate TSH, ACTH, and PRL secretion in vivo, although effects on GH levels were not detected (104). Interestingly, these inhibitory effects seem to occur through hypothalamic pathways, since production of corresponding releasing hormones by hypothalamic neurons was also decreased after in vitro treatment of medial basal hypothalamic fragments with thymosin-1 (105).

In addition to affecting the hypothalamus-pituitary axis, thymic hormones may act directly on its target endocrine glands (Fig. 5). In vitro experiments showed that thymulin can modulate gonadal tissues. Proliferation of oogonia from fetal rat ovaries, as well as gonocytes from fetal rat testicles, was consistently increased in the presence of thymulin (106, 107, 108). At least regarding the expansion of male germ cells in the same culture system, thymulin-inducing proliferative effects were largely prevented by TGF-ß1 (109).

View larger version (26K): [in this window] [in a new window]   Figure 5. Effects of thymulin on endocrine glands, as exemplified by the stimulatory effects on PRL, GH, TSH, and LH secretion by pituitary cells, as well as gonocyte proliferation.

It should be pointed out that the existence of direct effects of thymic hormones upon other endocrine glands that are physiological targets of the hypothalamus-pituitary axis, such as thyroid and adrenals, has not yet been studied.

Recent work has shown that thymulin can also modulate some peripheral nervous sensory functions, such as those related to pain sensitivity. In vivo injections of thymulin at high doses significantly reduced the hyperalgesia (related to both mechanical and thermal nociceptors) induced by intraplantar injection of endotoxin in rats and mice (113). Interestingly, when applied at much lower doses, this peptide instead generated hyperalgesia, an effect paralleled by a significant enhancement in the intrahepatic production of IL-1ß (114). Such paradoxical effects are in keeping with previous data showing that low doses of thymulin enhance IL-1ß secretion by peripheral blood cells, whereas, at high concentrations, thymulin suppresses its release as well as that of IL-2, IL-6, and TNF- (115). More recently, the cellular and molecular mechanisms involved in the thymulin-induced hyperalgesic effect have been further investigated. In the peripheral nervous system, the involvement of capsaicin-sensitive primary afferent neurons has been revealed. Intraperitoneal injection of capsaicin (known to destroy afferent nervous fibers) significantly abrogated the stimulatory thymulin effect on pain (116). Additionally, spinal cord neurons appear to be involved, since thymulin induces sustained expression of c-fos (a marker of spinal cord neuron activation) in those neurons known to be involved in nociception (117). It should be noted, however, that, in spite of these convincing data, the molecular basis for thymulin action on neurons is not complete, since thymulin receptors in neurons have not yet been determined.

Some data also indicate that other thymic hormones may exert a modulatory role in the central nervous tissue, including an effect on behavioral functions. In vivo injection of a thymopentin analog in rats was shown to counteract the stress response to experimentally induced social defeat (118), as measured in the elevated plus-maze apparatus, a recognized animal model of anxiety (119). Although the mechanism(s) involved in this thymopentin-mediated event were not characterized, direct action of the thymic hormone analog on the cholinergic innervation of the hypothalamus with consequent inhibition of CRF release was suggested (118). Such a putative direct anxiolytic effect of the thymopentin analog is further supported by previous data showing that injections of thymopentin normalize the numbers of benzodiazepine and -aminoisobutyric acid receptors in the hippocampus after stress (120). It is also noteworthy that neonatal thymectomy modulates the densities of nicotinic cholinergic receptors in skeletal muscle and brain (121).

For further details on the relationship between thymic hormones and the neuroendocrine system, including behavioral adaptive responses, see Refs. 122, 123 .

	V. Proliferation of Thymic Cells Is Hormonally Influenced

Top Abstract I. Introduction II. The Thymic Microenvironment... III. Neuroendocrine Control of... IV. Thymic Endocrine Function... V. Proliferation of Thymic... VI. Hormonal Modulation of... VII. Is Thymocyte Traffic... VIII. Expression of Receptors... IX. Intrathymic Expression of... X. Conclusions and Major... References

 
A. In vitro effects of pituitary hormones on TEC growth
One potentially relevant factor for better understanding thymus ontogeny is whether the expansion of TEC can be under neuroendocrine control. Pituitary hormones, such as PRL and GH, as well as IGF-I (78, 84), consistently enhance TEC proliferation in vitro. Similar results were obtained by such various methodological strategies as direct cell counting, [³H]thymidine incorporation, and immunofluorescence analysis of cultured TEC after bromodeoxyuridine uptake (Fig. 5, top panel). The proliferative effects of GH and IGF-I were recently confirmed using TEC lines derived from normal and from thymomatous rat thymus (124). Interestingly, the GH releasing inhibitor somatostatin, as well as its analog octreotide, significantly blocks human cultured TEC proliferation, as ascertained by [³H]thymidine incorporation (125).

A recent work suggested that TEC growth might also be under the control of thymic hormones. It was shown that thymopentin induces DNA synthesis in human TEC lines, either alone or in conjunction with FCS (126). Further studies using the complete thymopoietin molecule, in conjunction with similar assays using other thymic hormones, are necessary, however, to establish the concept of thymic hormone control of TEC growth.

Data are also scarce concerning the effects of steroid hormones on TEC proliferation. An apparent decrease in the proliferation rate was seen in a rat TEC line after treatment with progesterone, estrogens, or androgens, an effect probably mediated by protein kinase C (127). A similar inhibitory effect was seen with cortisol (128).

Much less is known about the in vivo effect of these hormones on TEC growth. However, it was shown that injections of metaclopramide, which promotes hyperprolactinemia, increased the number of solid epithelial islands in adult rat thymuses (129).

In vivo, Scheiff and co-workers (130) provided morphometric evidence that thyroid hormones were also able to induce TEC proliferation. Nevertheless, we and others observed no significant in vitro growth effect of T₃ using the model of a murine TEC line (66, 131). The reason for these apparently contrasting results is likely to lie in the distinct evaluation methodologies as well as the in vivo vs. in vitro situations.

B. Modulation of thymocyte proliferation by hormones and neuropeptides
In vitro thymocyte proliferation can be stimulated by supernatants of TEC cultures, whereas supernatants derived from fibroblast cultures have no effect. This thymocyte proliferative activity of TEC-derived supernatants was almost completely abolished by the presence of antithymulin monoclonal antibodies, but was enhanced when TEC were treated with T₃. By contrast, in the same study, T₃ did not directly influence thymocyte proliferation, as ascertained by short-term thymidine incorporation (132).

More recently, similar proliferative effects were seen using supernatants from PRL- or GH- treated TEC cultures (133, 134). Additionally, as depicted in Fig. 6, bottom panel, GH itself synergized with anti-CD3 in its stimulatory effect on thymocyte proliferation (135). This is in keeping with recent data showing that transgenic mice that overexpress GH or GH releasing hormone exhibit overgrowth of the thymus (136).

View larger version (56K): [in this window] [in a new window]   Figure 6. In vitro modulation of TEC and thymocyte proliferation by hormones. In the top panel, the stimulatory effect of PRL on human TEC growth is ascertained by direct cell counting, [3H]thymidine incorporation, and bromodeoxyuridine uptake, revealed by immunocytochemistry. The comitogenic effect of GH on thymocyte proliferation is seen in the bottom panel. In this experiment, mitogenic response was induced with anti-CD3 monoclonal antibody, and GH was added at various molar concentrations. Asterisks indicate statistically higher thymocyte expansion induced by anti-CD3 in the presence of GH. [Derived from Refs. 78 and 135.]

Further hormonal mediators are involved in the general control of thymocyte proliferation. For example, mitogen-induced human thymocyte expansion was blocked in the presence of anti-LH-RH serum, suggesting that intrathymically produced LH-RH may act as a costimulatory factor for thymocyte growth (140). At least in some experimental conditions, PRL also appears to be effective in directly stimulating thymocyte proliferation by enhancing IL-2 production and IL-2 receptor expression (141). This is keeping with data suggesting that PRL may play the role of a T cell growth factor, since it induces gene expression of cyclins D2 and D3 in the rat thymic lymphoma cell line Nb2 (142).

Proliferation of cultured human thymocytes was also directly stimulated in vitro by met-enkephalin, whereas VIP promoted an inhibitory effect, as compared with control untreated cultures, in both conditions of spontaneous and of phytohemagglutinin-induced mitogenesis (143). Similarly, somatostatin prevented concanavalin A-induced rat thymocyte proliferation (144). Another neuropeptide, CGRP, also inhibited mitogen-induced thymocyte proliferation, an effect that was abrogated in the presence of its antagonist CGRP8–37 (145).

Using the model of fetal thymus organ cultures, we recently demonstrated that expansion of thymocytes could be stimulated by insulin (146). Yet, since this is a complex heterocellular model, it remains to be determined whether the proliferative insulin effects are direct on the thymocytes or are mediated through microenvironmental cells. Tables 2 and 3 summarize the vast series of in vitro experiments concerning modulation of TEC or thymocyte proliferation by hormones and neuropeptides.

Injection of T₃ also promoted a consistent increase in thymocyte numbers with enhancement of spontaneous ex vivo [H³]thymidine incorporation (66).

Administration of synthetic TRH was also shown to enhance bromodeoxyuridine uptake by thymic cell suspensions (152). More recently, the same group showed in the rat model that such an effect could be seen in vivo, provided TRH was injected in the morning. In addition to confirming the previous data, these results reflect a time of day dependency for a physiological effect of TRH on thymocyte proliferation (153). Female rats continuously treated with leuprolide, an LH-RH agonist, exhibited a consistent increase in thymus weight (154), while in vivo injections of met-enkephalin in mice also enhanced thymus weight and cellularity (155). The latter finding is in keeping with the above in vitro stimulatory effect of this opioid on thymocyte proliferation.

Finally, intrathymic implants of the pineal gland derived from young mice, into age-matched recipients, led to a remarkable long-term maintenance of thymic size and the cortico-medullary architecture in the latter, thus preventing physiological age-related thymus atrophy (156). This suggested an effect of melatonin, either favoring proliferation and/or partially preventing apoptosis. In keeping with these findings, melatonin injection partially prevented stress-induced thymic atrophy (157). More recently, the mechanism by which melatonin could exert such an antiapoptotic role was revealed, with the demonstration that melatonin down-regulates the expression of glucocorticoid receptor (158).

By contrast, hormones such as sex steroids appear to provide an inhibitory tonus on thymocyte proliferation (reviewed in Ref. 159). Castration in young adult male mice promoted a rapid wave of thymocyte proliferation in vivo, particularly in cortically located cells bearing the immature phenotypes CD4^-CD8^- and CD4⁺CD8⁺ (160). This is in keeping with previous findings that castration in old rats led to enhancement of thymus weight, an effect that was abrogated by androgen treatment of castrated animals (161).

Taken together, the data summarized above clearly indicate that distinct hormones and neuropeptides can convey positive and negative signals for thymocyte proliferation.

	VI. Hormonal Modulation of Intrathymic T Cell Differentiation

Top Abstract I. Introduction II. The Thymic Microenvironment... III. Neuroendocrine Control of... IV. Thymic Endocrine Function... V. Proliferation of Thymic... VI. Hormonal Modulation of... VII. Is Thymocyte Traffic... VIII. Expression of Receptors... IX. Intrathymic Expression of... X. Conclusions and Major... References

 
A. Thymocyte-derived cytokine profile
One thymocyte function influenced by neuroendocrine circuits is the cytokine profile produced by these cells. For instance, concanavalin-A mitogenic response and IL-6 production were enhanced in thymocytes from GH-treated aging animals (162), and PRL induced IL-2 production by thymocytes (141). Vasopressin can replace IFN- in its role of inducing IL-2 production (163), and substance P stimulated IL-2 synthesis by the mouse thymic lymphoma cell line EL-4 (164). By contrast, VIP, PACAP27, and PACAP38 exerted an in vitro inhibitory action on the production of some thymocyte-derived cytokines, including IL-2, IL-4, and IL-10 (165, 166). Altogether, these findings provide consistent evidence for a hormone/neuropeptide balance in the control of cytokine production by thymocytes.

B. Changes in the T cell differentiation markers and TCR Vß repertoire
As detailed above, hormones and neuropeptides can affect thymic functions related to thymocyte differentiation. Thus, such differentiation could also be a target for neuroendocrine control. Implants of GH3 pituitary cells in aging rats increased total thymocyte numbers and the percentage of CD3-bearing cells, with a parallel decrease in the CD4^-CD8^- double-negative thymocytes, which normally accumulate in the aging rat thymus (147, 167). The role of GH in thymus development was further supported by findings in GH-deficient dwarf mice. In addition to the precocious decline in thymulin serum values (80), there was progressive thymic hypoplasia with decreased numbers of CD4⁺CD8⁺ double-positive thymocytes. Such defects could be restored by prolonged treatment with GH (168).

Injections of T₃ promoted an increase, both in relative and absolute numbers of thymocytes bearing the CD44 marker (66), which is an ECM receptor of the proteoglycan family, with specificities for hyaluronate and to a lesser extent fibronectin and collagen. By contrast, administration of high doses of glucocorticoid hormones yielded a profound decrease in the percentages of CD4⁺CD8⁺ thymocytes, with a relative increase in CD4^-CD8^- as well as CD4⁺CD8^- and CD4^-CD8⁺ cells (169, 170). In vivo treatment of mice with estradiol also promoted a depletion in the absolute numbers of CD4⁺CD8⁺, CD4⁺CD8^-, and CD4^-CD8⁺ thymocytes, with a decrease in the proportion of double-positive cells and an increase in the percentage of double-negative as well as single-positive mature cells (171). A striking loss of the very immature CD3^-CD4^-CD8^- cells was further demonstrated (172). Interestingly, the estrogen-induced thymic involution appeared to occur independently of glucocorticoids, since it was seen in adrenalectomized animals (170). In this same study, the proportions of CD4⁺CD8⁺ thymocytes in adrenalectomized estrogen-treated mice, although significantly lower than in control animals, were higher than those in hormone-treated mice not subjected to adrenalectomy.

Although considerable work is available concerning the neuroendocrine control of T cell differentiation markers, the influence of hormones and neuropeptides on shaping the intrathymically generated T cell repertoire remains poorly studied. However, the few data available point to such an influence. Mice treated with estradiol exhibited a selective increase in the percentages of CD4^-CD8^- TCR⁺ thymocytes expressing Vß6, Vß8, or Vß11 but not Vß3 gene products (173), thus promoting an imbalance in the generation of the TCR repertoire of the double-negative TCR⁺ cell lineage. Interestingly, an enhancement of IL-1 mRNA was seen in parallel with the increase in Vß8⁺ cells, in keeping with previous data showing that this cytokine exerts a mitogenic effect on Vß8⁺ thymocytes (173). More recently, release of autoreactive T cells bearing the Vß3 or Vß11 phenotypes, with autoreactivity to hepatocytes, was seen in mice injected with a single dose of estradiol (174).

In rat lymphoma-derived Nb2 cells, PRL induced in vitro gene expression of the TCR chain, whereas the TCR ß chain gene was suppressed (175), suggesting that the intrathymic PRL content may drive T-cell differentiation pathways. In keeping with this hypothesis, it has been demonstrated, not only in the Nb2 cell line but also in human thymocytes, that in the absence of TCR ligation PRL induces rapid phosphorylation of multiple TCR/CD3 complex proteins, including the CD3 -chain and the ZAP-70 tyrosine kinase, both essential for TCR function (176). However, recent data argue against the above hypothesis, since PRL receptor knockout mice apparently develop a normal thymocyte differentiation pathway, at least in terms of CD3, CD4, and CD8 markers (177, 178).

As detailed below, very elegant data strongly suggest that intrathymically produced glucocorticoids influence the generation of the T cell repertoire in the thymus by modulating positive and negative selection of thymocytes.

C. Hormone-mediated apoptosis in thymocytes
One of the best studied effects concerning the hormonal control of intrathymic cell death is that mediated through glucocorticoid hormones. When applied in relatively high doses, these substances trigger an apoptotic cell machinery in differentiating thymocytes, particularly cells bearing the immature CD4⁺CD8⁺ phenotype. The molecular basis of such a glucocorticoid hormone-induced biological response is now known in detail and is used as a paradigm for studies of apoptosis in various cell types (reviewed in Ref. 179). Briefly, glucocorticoids activate calcium-dependent endonucleases that eventually cleave DNA, with the formation of oligo-nucleosomes. A series of findings suggest that such events are potentiated by cAMP (180) and depend on the recently isolated transcription factor SRG3 (181). They do not require p53 tumor suppressor gene expression, which has been shown to be necessary for triggering apoptosis in thymocytes by contact with TEC or after radiation (182, 183). Oligonucleosomal DNA fragmentation is preceded by the enzymatic degradation of lamin B1, a protein involved in the nuclear lamina architecture (184). Interestingly, glucocorticoid-induced thymocyte apoptosis also modulates cytoplasmic processes, including thiol oxidation in mitochondria (185), activation of the protease calpain (186), and decrease in the intracellular K⁺ concentration (187). The need for glucocorticoids to bind to the corresponding receptor to induce apoptosis in thymocytes has been definitely established by the demonstration that thymocytes from glucocorticoid receptor null mice do not undergo apoptosis after the respective hormonal treatment (188). In a recent study, it was further shown that glucocorticoid-dependent apoptosis in thymocytes is mediated by transactivation of the glucocorticoid receptor (189). This was approached by constructing a point mutation in the glucocorticoid receptor gene, able to abolish DNA binding-dependent transactivation, and further generating mice expressing this mutant gene. Thymocytes from these animals were resistant to dexamethasone-induced apoptosis, thus showing that DNA binding of the glucocorticoid receptor is a prerequisite for glucocorticoid-induced apoptosis.

Although the mechanism of glucocorticoid-induced apoptosis in thymocytes is relatively well known at the molecular level, a paradox remains to be understood: low doses of glucocorticoid hormones antagonized TCR-mediated apoptosis in thymocytes (190, 191) and partially rescued thymocytes from apoptosis induced by in vitro treatment of fetal thymus organ cultures with anti-CD3 antibodies (192). As further discussed below, this may be relevant for a physiological role of these substances in positive selection of the T cell repertoire. Thus, understanding the molecular control of these balancing effects demands further investigation.

Although less studied, sex steroids also appear to affect thymocyte apoptosis. For example, injections of estradiol in rats promoted cortical thymocyte depletion (193), with an increase in apoptosis (ascertained by caryopyknosis) and a decrease in mitotic indexes (194, 195, 196, 197) (see Table 2).

A natural condition for studying the influence of sex steroids on thymocyte apoptosis is pregnancy, during which the thymus undergoes progressive and extensive involution with loss of CD4⁺CD8⁺ cells, partially due to apoptosis (198). Interestingly, this progressive thymic atrophy of pregnancy inversely correlates with the rise in circulating progesterone (199). Other hormones such as pituitary hormones and glucocorticoids could also be involved in this process.

Androgens, such as testosterone, also induce thymocyte death in vivo, by a mechanism independent of the glucocorticoid and estrogen receptors (200), but which requires specific androgen receptors since the effect is prevented by treatment with the antiandrogen drug flutamide. Since the levels of androgen receptor mRNA are 6-fold higher in TEC than in thymocytes, it was postulated that the induction of thymocyte apoptosis by androgens is mediated by TEC-derived products (200). The acceleration of thymocyte apoptosis by androgens was recently confirmed in vitro using thymic organ culture (201).

Studies in mice revealed that estrogen treatment also causes a dramatic reduction in thymic size and cellularity. All CD4/CD8-defined T cell subsets were reduced, particularly the CD4⁺CD8⁺ double positive cells. Examination of the CD3^-CD4^- CD8^- subset revealed a striking loss of developmental progression of the early precursor cells, since in treated animals, this compartment was composed almost entirely of the earliest population, CD44⁺CD25^-, with depletion of cells in the remaining maturational stages, CD44⁺CD25⁺, CD44^-CD25⁺, and CD44^-CD25^- (202). It should be recalled that in these studies, apoptosis was not specifically checked with any of the present available tools. Intriguingly, in the same work, the authors showed that estrogen deprivation by oophorectomy did not enhance T cell development. Such a paradox is further apparent in recent findings that estrogen receptor knockout mice exhibit lower thymic cellularity compared with normal or heterozygous littermates (203). In these animals, despite the reduced cell numbers, thymocyte maturation stages were apparently normal, as revealed by CD3, CD4, CD8, CD25, and CD44 labeling. Nevertheless, their sensitivity to in vivo estradiol treatment was distinct from wild or heterozygous age-matched counterparts. While estrogen receptor null mice underwent thymic atrophy with estradiol treatment (thus similar to control animals), the phenotypic differences in thymocyte subpopulations such as loss of CD4⁺CD8⁺ cells, seen in usual conditions of estradiol treatment, were not detected in mutant mice. These findings point to a dichotomy of estradiol in terms of influence on thymocyte numbers and differentiation. In fact, in the same work, chimera experiments elegantly demonstrated that the sensitivity to estradiol in terms of thymic atrophy was dependent on the expression of the null mutation in the thymic microenvironment rather than in thymocytes.

Protection from apoptosis may also be under neuroendocrine control. Dihydroepiandrosterone, for example, is apparently able to counteract at least in vivo thymocyte apoptosis induced by pharmacological doses of glucocorticoid hormones (204, 205).

Very few data are so far available concerning the role of pituitary hormones in modulating thymocyte apoptosis. Using the rat lymphoma Nb2 cell line, it was demonstrated that the apoptotic effect of dexamethasone was inhibited in a dose-dependent manner by PRL or GH (206), raising the hypothesis that a similar effect may occur with normal thymocytes. In fact, as regards PRL, this hypothesis fits with the data discussed above, showing that this hormone can trigger phosphorylation of TCR-related proteins (207).

The VIP also appears to play a role in protecting thymocytes from apoptosis induced by dexamethasone treatment in vitro. It inhibits the typical DNA fragmentation induced by glucocorticoids and increases cell survival, both effects being mediated through the VIP receptor (208).

The pineal neurohormone melatonin also appears to partially prevent apoptosis of rat thymocytes in vivo and in vitro, as defined by morphological criteria and DNA fragmentation seen in agarose gel electrophoresis (209). Similar findings were obtained in aging mice, where thymus atrophy was partially prevented by long-term melatonin treatment or pineal grafting (210). As mentioned above, this antiapoptotic effect of melatonin is likely to be, at least in part, due to down-regulation of the glucocorticoid receptor (158).

These data indicate that neuroendocrine-mediated regulation of thymocyte apoptosis does exist, facilitating or preventing the cells to enter a programmed pathway of cell death. They favor the notion that negative vs. positive selection of the T cell repertoire within the thymus may be under the control of a neuroendocrine homeostatic axis involving various hormones and neuropeptides. Consequently, it should be important to define to what extent such neuroendocrine circuits influence shaping of the intrathymically generated T cell repertoire.

	VII. Is Thymocyte Traffic Under Neuroendocrine Control?

Top Abstract I. Introduction II. The Thymic Microenvironment... III. Neuroendocrine Control of... IV. Thymic Endocrine Function... V. Proliferation of Thymic... VI. Hormonal Modulation of... VII. Is Thymocyte Traffic... VIII. Expression of Receptors... IX. Intrathymic Expression of... X. Conclusions and Major... References

 
A. Effects on the entrance of cell precursors into the thymus
Very few studies have been conducted so far to determine whether the entrance of T cell precursors into the thymus is under neuroendocrine control. It was demonstrated that recombinant human GH increases human T cell engraftment into the thymus of SCID mice (211). These investigators suggested that such influence is mediated by adhesion molecules and ECM, since the entrance of T cells into the thymus can be abrogated with anti-ß1 or anti-ß2 integrin antibodies, and GH-treated cells exhibit an increase in adhesion to ICAM-1, VCAM-1, and fibronectin (211). In keeping with this idea, it was recently demonstrated that administration of IGF-I together with bone marrow cells into syngeneic old recipients resulted in an increase in thymus cellularity, compared with transfer of bone marrow cells alone (212). In the same study, it was demonstrated that IGF-I potentiates the colonization of fetal thymus organ cultures with T cell precursors, clearly indicating that IGF-I enhances the entrance of cell precursors into the thymus.

A second example is dexamethasone and hydrocortisone enhancement of the in vitro migration of bone marrow-derived progenitor cells into the thymus (213). Additionally, in vivo treatment of rats with estradiol enhanced permeability of cortical blood vessels (214), which may facilitate the entry of pro-thymocytes. However, this issue has not yet been experimentally approached.

It has been reported that the lack of maternal glucocorticoids in the progeny of adrenalectomized pregnant rats accelerates early colonization of the thymic primordium by lymphoid progenitors (62, 63). As previously discussed herein, this is probably related to the earlier maturation of the thymic microenvironment seen in this particular experimental condition.

B. Modulation of thymocyte traffic in TNCs
Intrathymic lymphocyte traffic also appears to be under neuroendocrine control. Spontaneous as well as phorbol ester-induced in vitro mobility of thymocytes was shown to be inhibited by VIP and the two pituitary adenylcyclase-activating polypeptides (PACAP27 and PACAP38), an effect paralleled by a rise in cAMP concentration (215). Whether these neuropeptides change the expression of membrane receptors related to cell migration remains to be determined.

Considering that ECM plays a role in intrathymic cell migration (17), and since various hormones could up-regulate the expression of ECM ligands and receptors (56, 66), we investigated whether thyroid and pituitary hormones could be involved in TEC-thymocyte interactions related to cell migration. One key interaction is the heterotypic adhesion of thymocytes to epithelial cells. As discussed above, we demonstrated that these hormones up-regulate TEC-thymocyte adhesion, as ascertained by direct counting under the optical microscope or by ELISA using CD90 (the Thy.1 antigen) as a membrane marker for thymocytes (56, 66). With regard to PRL and GH, the hormonal specificity of this effect was confirmed since respective antihormone antibodies could block it, totally or partially. We also noted that the GH-enhancing effects were prevented by anti-IGF-I or anti-IGF-I receptor antibodies. The involvement of ECM-mediated interactions was further demonstrated since the hormone-enhancing effects on TEC-thymocyte adhesion were also abrogated by antibodies with specificities for fibronectin, laminin, and their respective receptors, VLA-5 and VLA-6 (56, 216).

We further attempted to modulate a direct cell migration process, namely the entrance of thymocytes into and their exit from TNCs. In a first set of experiments (Fig. 7), we showed that thymocyte release from TNC complexes was accelerated if these complexes were treated in vitro with T₃. Most importantly, when TNCs were harvested from T₃-treated mice, thymocyte release was also faster than in controls (66). More recently, we observed that when TNC-derived epithelial cells were treated with the thyroid hormones and cocultured with fetal thymocytes, the ratio of reconstitution of lymphoepithelial complexes was enhanced (our unpublished results), indicating that the entrance of thymocytes into TNC is also hormonally regulated. Further in vitro studies revealed that both thymocyte release from TNC and TNC reconstitution were consistently increased if cultures were subjected to PRL, GH, or IGF-I (56).

View larger version (47K): [in this window] [in a new window]   Figure 7. Enhancement of thymocyte release from mouse TNC complexes by T3. The upper panels show schematically the three stages of TNC complexes: round TNCs that did not release thymocytes (left), spread epithelial cells that partially released thymocytes (middle), and thymocyte-free epithelial cells (right). The middle panels depict thymocyte release from TNCs derived from control () or T3-injected mice () and the bottom panels, TNCs recovered from normal mice and treated in vitro with 10-8 M () or 10-10 M () of T3. After both in vivo and in vitro treatment, thymocyte release was significantly faster (*P < 0.01) than in corresponding controls. [Derived from Ref. 66.]

One direct strategy to evaluate thymocyte exit in adult animals is analysis of the so-called recent thymic emigrants. It is well established that intrathymic injection of fluorescein isothiocyanate (FITC) randomly labels the cell membranes of many thymocytes. This allows recovery of the FITC⁺ cells that have recently exited the thymus (217, 218). We observed, 16 h following a single intrathymic injection of T₃, a consistent increase in the numbers of FITC⁺ cells in lymph nodes (219). Additionally, using a similar protocol, we noted that GH is also able to modulate the homing of recent thymic emigrants, enhancing the numbers of FITC⁺ cells in the lymph nodes and diminishing them in the spleen (220).

An in vivo model in which immature CD4⁺/CD8⁺ T cells were detected in peripheral lymphoid organs (thus reflecting an abnormal thymocyte exit) is that of transgenic mice with impaired corticosterone receptor function by partial knockout of the glucocorticoid receptor, secondary to endogenous expression of the corresponding antisense RNA (221). These animals showed disruption of the hypothalamus-pituitary-adrenal axis, bearing abnormally high circulating levels of ACTH and corticosterone (222). The intrathymic levels of glucocorticoid receptor mRNA were 50% lower, and the ability of thymus extracts to bind hydrocortisone was reduced to one third of that in normal mice (221). In this respect, it is interesting to note that intrathymic expression of the antisense was restricted to thymocytes since the construct applied used the promoter from the thymocyte-specific thyrosine kinase lck.

Abnormal leakage of CD4⁺CD8⁺ cells was also detected in lymph nodes of the Snell Bagg dwarf mouse (168). This phenomenon progressed with age and paralleled the cortical thymocyte depletion found in dwarf mice. In those animals with dramatic loss of CD4⁺CD8⁺ thymocytes, the percentages of CD3⁺CD4⁺CD8⁺ cells in the lymph nodes reached 50%. Importantly, both CD4⁺CD8⁺ thymocyte depletion and the abnormal presence of CD4⁺CD8⁺ cells in the periphery were restored after daily GH injection (168).

Lastly, it is noteworthy that release of immature CD4⁺CD8⁺ thymocytes may also be under the influence of sex hormones. Adult rats treated with estradiol benzoate exhibited CD4⁺CD8⁺ in the spleen, which was related to increased vascular permeability of cortical blood vessels (223).

Taken together, the three models summarized above indicate that a disturbance in distinct neuroendocrine compartments may result in a partial imbalance of the process of thymocyte exit, allowing immature cells to emigrate from the organ. Considering the autoreactive potential of CD4⁺/CD8⁺ cells found in peripheral lymphoid organs from neonates of certain mouse strains (224), it is conceivable that a neuroendocrine-driven imbalance resulting in abnormal traffic of immature thymocytes toward peripheral lymphoid organs may favor the development of some autoimmune diseases.

It is worth emphasizing that the question of whether the neuroendocrine control of thymocyte migration occurs partially through the modulation of thymic microenvironment-derived chemokines has not been tested in any model and certainly represents a promising field for original investigation.

	VIII. Expression of Receptors for Hormones and Neuropeptides by Thymic Cells

Top Abstract I. Introduction II. The Thymic Microenvironment... III. Neuroendocrine Control of... IV. Thymic Endocrine Function... V. Proliferation of Thymic... VI. Hormonal Modulation of... VII. Is Thymocyte Traffic... VIII. Expression of Receptors... IX. Intrathymic Expression of... X. Conclusions and Major... References

 
The above discussion of the variety of effects of distinct hormones and neuropeptides on thymus physiology obviously implies that distinct thymic cell types should express receptors for such molecules. As detailed below, the expression of specific receptors for adrenal and sex steroids, thyroid and pituitary hormones, as well as neuropeptides has been detected by several groups, using various methodological approaches, not only in thymocytes but also in TEC (Tables 2 and 4).

Receptors for sex steroids have also been demonstrated in animal and human thymus. Pioneer studies by Grossman and co-workers (236, 237, 238) showed, by binding assays, that specific receptors for estrogen, progesterone, and androgen are present particularly in thymic preparations obtained from nonlymphoid cells. Other groups confirmed the presence of androgen and progesterone receptors in TEC-enriched fractions (239, 240, 241, 242). In one study, a quantitative RT-PCR approach for detecting androgen receptor mRNA in the thymus showed 6-fold more mRNA in the TEC than in the thymocytes (243). Immunocytochemically based flow cytometry analysis of freshly isolated thymocytes revealed androgen receptor expression in all classes of CD4/CD8-defined thymocyte subsets, with the highest levels observed in the CD4^-CD8^- subset, including the most immature cells (244).

With respect to progesterone receptor, direct binding of peroxidase-coupled ligands onto thymus sections (245), as well as immunocytochemistry, revealed the colocalization of progesterone receptor-positive and estrogen receptor-positive cells with TEC-specific cytokeratin staining (246).

The expression of estrogen receptor in thymocytes was determined though binding assay (247), as well as by immunoblotting and flow cytometry (248). According to previous histochemical analyses obtained with human thymic specimens, the binding is located in the reticulo-epithelial stroma rather than the thymocytes (249).

More recently, a novel form of the human estrogen receptor, the ERß form, was cloned, and the corresponding mRNA was found in the thymus (250).

Initial observations indicated that thymocytes exhibit specific binding sites for thyroid hormones (251). Nuclear T₃ receptors (NT₃-R) were identified in both TEC and thymocytes by immunochemical approaches using a specific anti-NT₃-R monoclonal antibody, revealing a 57-kDa protein similar to the NT₃-R originally described in the liver. Although flow cytometry analysis defining the expression of NT₃-R as a function of the degree of thymocyte differentiation is still lacking, in situ immunocytochemical labeling clearly showed that most thymocytes express this receptor (252).

Expression of NT₃-R by the murine thymic epithelium was also determined in various TEC preparations including TNC and TEC lines. Interestingly, NT₃-R expressed by TEC seems to be functional since the epitope recognized by the anti-NT₃-R monoclonal antibody (mAb) was transiently down-regulated by treatment of TEC cultures with T₃ (252). More recently, the presence of NT₃-R was also demonstrated in cultured human TEC (our unpublished data).

B. Expression of PRL and GH receptors by thymocytes and TEC
Receptors for various pituitary hormones have also been defined in distinct thymic cell types. Taking the PRL receptors as an example, we first showed that they are expressed on TEC in situ and in vitro, as defined by immunocytochemistry, immunoblotting, and Northern blotting. Their functional activity was evidenced by modulation of both thymulin production and TEC growth with appropriate agonistic doses of anti-PRL receptor antibodies (253). Additionally, PRL receptors were expressed by most thymocytes, independently of their CD4/CD8-defined maturation stage. The receptor density on cell membranes was enhanced after in vitro concanavalin A mitogenic stimulation (254, 255). Interestingly, high levels of circulating PRL, as occur in lactation, enhance the levels of PRL receptors in thymic cells, as revealed by binding assay and semiquantitative RT-PCR (256).

Expression of GH receptor by cultured human TEC was initially shown by means of binding assay (257) and more recently by immunocytochemistry and RT-PCR (258). This is in keeping with in situ immunocytochemical data in the avian thymus, showing colocalization of the GH receptor with epithelial cells, defined by cytokeratin labeling (259). Additionally, in situ hybridization studies in the rat thymus suggested epithelial labeling for the GH receptor mRNA (260).

GH receptor expression in murine thymocytes was particularly seen in the CD4^-CD8^- immature subset (261). Similar findings were made in human thymocytes in which GH receptor expression was mainly restricted to the very immature differentiation stage defined by the phenotype CD34⁺CD2⁺CD3^-CD4^-CD8^- (258), suggesting that the direct effects of GH on thymocytes precede events related to the selection of the T cell repertoire. It should be noted, however, that in bovine fetal thymus, GH receptor was found in both CD4⁺ and CD8⁺ single positive thymocytes (262). Such differences can be ascribed to species specificity, the fact that the latter study was conducted in early stages of thymus development, and/or sensitivity to the distinct reagents used for detecting the GH receptor by flow cytometry.

Both the receptor and GH binding proteins were found in the thymus of mammalian and avian thymuses (259, 260).

C. Receptors for neuropeptides in thymic cells
Intrathymic expression of receptors for various neuropeptides has been shown in thymocytes, with fewer studies carried out in TEC and other components of the thymic microenvironment. This is the case for oxytocin and vasopressin receptors, determined on thymocyte-derived membrane preparations by means of ligand binding and PCR (263, 264). GH-RH receptors have also been shown in rat thymocytes by means of binding assay and direct coupling of the radioactive ligand onto electrophoresed thymocyte-derived extracts, revealing two protein bands (43 and 27 kDa) similar to those in pituitary gland-derived preparations (265), whereas the TRH receptor has been shown in thymus extracts by RT-PCR (266). The somatostatin receptor family has been identified in mouse thymocytes, as ascertained by gene expression of the receptor subtypes SSTR2A, SSTR2B, and SSTR3 (267). Autoradiographic analysis of radioactive ligands directly bound to human thymus sections indicated that the family is also present in TEC (268). This was confirmed by various methodological strategies including binding assay and RT-PCR (125). It was also demonstrated that somatostatin receptors in human TEC are functional since both somatostatin and its analog octreotide significantly inhibited in vitro the proliferation of human TEC (125).

Receptors for endogenous opioids such as ß-endorphin and met-enkephalin have been shown by means of binding assays on membrane preparations from total thymus extracts (269). These findings confirmed previous data using the same methodological approach, revealing opioid receptors on thymocyte membranes (270). The presence of such receptors in thymic microenvironmental cells remains to be determined.

The expression of receptors for other neuropeptides has also been demonstrated in the thymus. For example, the mRNA coding for the VIP₁ receptor was detected in thymocytes (271), particularly those bearing the most immature phenotype, the CD4^-CD8^- cells (272). Interestingly, if the expression of VIP₁ receptor is constitutive, thymocytes can express the VIP₂ receptor only after mitogenic stimulation with anti-CD3 antibodies (215). More recently, VIP1 receptor has been identified in human TEC by means of immunocytochemistry, cytofluorometry, RT-PCR, and Southern blotting (273). This receptor is functional since it stimulates cAMP production by cultured TEC, which is keeping with the in situ radiolabeling detection of VIP receptor in both cortex and medulla of the human thymus (274).

Receptors for other members of the VIP family, such as the PACAP peptides, have been identified by binding assay (275), although the same research group did not detect PACAP 27 binding sites in thymus autoradiographs (276). Binding sites for CGRP were also detected in thymocyte membranes (145); their presence in the thymic microenvironment has not been studied so far. More recently, the CGRP receptor was demonstrated biochemically and functionally in human TEC (273). Finally, melatonin-binding sites have been detected in thymocyte preparations as well (277, 278).

	IX. Intrathymic Expression of Hormones and Neuropeptides

Top Abstract I. Introduction II. The Thymic Microenvironment... III. Neuroendocrine Control of... IV. Thymic Endocrine Function... V. Proliferation of Thymic... VI. Hormonal Modulation of... VII. Is Thymocyte Traffic... VIII. Expression of Receptors... IX. Intrathymic Expression of... X. Conclusions and Major... References

 
The examples cited above clearly indicate that a classical endocrine pathway is partially implicated in various effects of hormones and neuropeptides on thymic lymphoid and microenvironmental compartments and that target cells are triggered via specific receptors. Nevertheless, another point deserving discussion in respect to neuroendocrine control of the thymus is related to the intrathymic production of several hormones and neuropeptides.

A. Intrathymic production of corticosterone: role in the shaping of the T cell repertoire
Pioneer work by Ashwell and colleagues (279) revealed that not only do TEC bear the enzyme machinery necessary for corticosteroid biosynthesis, but, at least in the murine thymus, corticosterone is actually produced by TEC. More recently, two independent research groups confirmed these results. Additionally, by transfecting a target cell with a plasmid containing two glucocorticoid-responsive elements plus a luciferase reporter gene, the authors showed that secretion of TEC-derived glucocorticoids was enhanced by ACTH and blocked by the glucocorticoid receptor antagonist RU486 as well as two blockers of corticosterone biosynthesis, trilostane and metyrapone (280). Interestingly, in a further recent study, it was shown that, although the whole set of steroidogenic enzymes and cofactors for the synthesis of glucocorticoids could be detected in murine thymic tissue, intact thymic architecture was necessary for glucocorticoid production, since 11ß-hydroxylase was not detected in irradiated thymus or in a TEC line (281).

Since low or moderate doses of glucocorticoids were found to prevent anti-CD3-induced apoptosis, it was hypothesized that thymus-derived glucocorticoids might play a physiological role in positive selection of the T cell repertoire. Addition of metyrapone (a selective inhibitor of corticosteroid biosynthesis) to fetal thymus organ cultures enhanced TCR-mediated thymocyte deletion, an effect that could be reversed by exogenous addition of corticosterone to the cultures (279). More recently, using the same model of fetal thymus organ cultures from mice bearing transgenic ßTCRs, it was shown that thymus-derived glucocorticoid hormones prevent thymocyte apoptosis only when the TCR is capable of recognizing the self-antigen/MHC complex with sufficient avidity to normally undergo positive selection (192). In a second vein, transgenic mice expressing antisense transcripts for the glucocorticoid receptor exhibited thymic atrophy particularly due to CD4⁺CD8⁺ cell depletion and enhanced susceptibility to TCR-mediated apoptosis (282). When this transgene is transferred into the MRL mice that spontaneously develop autoimmunity, there is a decrease in the TCR-Vß bearing cells that are otherwise positively selected, and such animals then exhibit lower autoantibody production and milder symptoms of the autoimmune disease (283).

Although dealing with transgenic animals, these findings indicate that, under normal conditions, endogenous glucocorticoids might prevent thymocyte apoptosis after TCR/peptide-MHC interaction. In fact, similar data were recently reported with nontransgenic normal syngeneic mice (284).

However, this issue deserves further investigation, since in other studies, it was found that RU 486, an antagonist of the glucocorticoid receptor, blocks anti-CD3 induced apoptosis in newborn-derived thymus organ cultures (285). Additionally, in vivo treatment with RU 486 in mice transgenic for one particular TCR able to recognize ovalbumin also prevented thymocyte apoptosis induced by specific antigen stimulation (286).

Whether other steroid hormones, including sex steroids, are produced intrathymically remains to be determined. This is a potentially important issue, given the well established sexual dimorphism of the immune response in normal and autoimmune conditions.

B. Expression of "classic" adenopituitary hormones by thymic cells
The expression of PRL and GH by cells of the immune system, including the thymus, has been extensively documented. Since most of the data were recently reviewed (287), it will be only briefly discussed herein.

PRL gene expression has been detected by distinct research groups in thymocyte-derived preparations (288, 289). The detection of a PRL-immunoreactive molecule (290, 291) indicates that thymocytes constitutively produce PRL. However, the possible roles of thymus-derived PRL and of microenvironmental cells in intrathymic PRL production remain to be investigated.

Intrathymic GH expression was defined by detection of the corresponding mRNA by in situ hybridization and the peptide by immunocytochemistry (292); positive signals were revealed in cortical epithelial cells and in septal phenotypically-undefined cells, but not in thymocytes. This contrasted with previous detection of GH mRNA and corresponding protein in rat thymocyte-derived preparations (265, 293), and with the detection of an immunoreactive, 22-kDa, biologically active GH from human thymocytes isolated ex vivo (137). More recently, we showed the production and secretion of GH by human thymocytes isolated ex vivo as well as in primary TEC cultures, using RT/PCR and immunoradiometric assays. GH gene expression by rat thymocytes is up-regulated by GH-RH (265), in keeping with the demonstration of Pit-1/GHF-1 transcription factor (which controls GH expression in the pituitary gland) in human thymic microenvironmental cells (294). Nevertheless, it should be mentioned that, as yet, no experimental data are available concerning whether GH production and release by distinct thymic cell types are under the same Pit-1/GHF-1-dependent control mechanism in normal conditions. Studies in dwarf mice showed that GH expression by thymocytes and bone marrow microenvironmental cells does not depend on Pit-1 (295, 296). From a functional point of view, it was found that thymocyte-derived GH can enhance thymocyte proliferation through an IGF-I-mediated circuit (137).

If the production of PRL and GH by thymic cells is well documented, evidence for intrathymic expression of other adenopituitary hormones is still scarce. An immunoreactive LH peptide was extracted from human thymocytes (140), and it was shown that PHA-stimulated mitogenic response of these cells was blocked in the presence of anti-LH antiserum. Additionally, immunocytochemical findings indicate that TSH, FSH, and ACTH may be produced within the thymus, particularly by the epithelial component (297, 298). The POMC gene also appears to be constitutively expressed in the thymus, as ascertained by Northern blotting, RT-PCR, and immunocytochemistry (299, 300, 301). The latter study, performed in the chicken thymus, indicated the presence of POMC in TEC and dendritic cells.

Despite the findings discussed above, further studies are needed to define whether these pituitary hormones are actually secreted intrathymically and how they are regulated. It is interesting that the degree of intrathymic hormone production can be disrupted in some pathological conditions, as exemplified by the high quantities of ACTH detected in some carcinoid tumors (302).

C. Is there a functional autocrine/paracrine IGF-I-mediated circuitry in the thymus?
As partially discussed above, converging data now strongly indicate that IGF-I is involved in the effects of GH on the thymus (reviewed in Ref. 287): 1) control of thymulin secretion and TEC/thymocyte adhesion by GH can be prevented in vitro by treating TEC cultures with monoclonal antibodies specific for IGF-I or IGF-I receptor (56, 84); and 2) IGF-I alone can replace GH in stimulating thymulin production by cultured TEC and in increasing TEC/thymocyte adhesion (56, 84). Moreover, the enhanced concanavalin-A mitogenic response and IL-6 production by thymocytes observed in GH-treated aging animals (162) can be detected in animals treated with IGF-I (303). In the same vein, it was shown that cyclosporin A-induced thymic atrophy was restored by in vivo treatment with recombinant GH or IGF-I (148), and that IGF-I was able to induce repopulation of the atrophic thymus from diabetic rats (304). Taken together, these findings strongly suggest that GH triggers or enhances a functional circuitry in the thymus involving IGF-I and its receptor, thus implying the intrathymic production of these molecules. Recent findings showing that exogenous or thymus-derived GH promotes thymocyte proliferation via IGF-I production by these cells are noteworthy (137, 305). This is in keeping with the demonstration of IGF-I receptors in thymocytes (306, 307).

In a second vein, we recently provided immunochemically based evidence regarding the expression of IGF-I and IGF-I receptor by murine and human TEC, as ascertained by immunocytochemistry, immunoblot, and immunodot analyses. Both molecules are constitutively expressed by TEC, but their densities can be increased after GH treatment (our unpublished data). However, recent findings in experiments using ribonuclease protection assay in human TEC extracts failed to detect mRNA for the IGF-I receptor (308). At present, we cannot explain such contrasting results. They may be due either to the methodology or to the samples used for running the various assays.

In any case, it remains plausible that GH up-regulates a functional IGF-I/IGF-I receptor circuitry in both lymphoid and microenvironmental compartments of the thymus. However, the relative contribution of thymus-derived vs. pituitary gland-derived GH to the regulation of IGF-I and its receptor in thymocytes or TEC is unknown at the moment.

IGF-I is not the only member of the insulin family expressed in the thymus. Immunocytochemical evidence suggests that IGF-II may be much more expressed intrathymically than IGF-I (309). Considering that IGF-II receptors have been shown in the rat and the human thymus (306, 308, 310), together with the demonstration of various IGF binding proteins in this tissue (308), the existence of an intrathymic biological circuit mediated by IGF-II and its receptor is also conceivable.

Insulin expression has also been demonstrated in the human thymus (311, 312) and confirmed in the mouse along with other pancreatic hormones (313, 314). Particularly in the mouse, we showed that insulin expression is restricted to thymic dendritic cells (313). This was further confirmed by RT-PCR in studies using FTOC, in which insulin gene expression was not found in MHC class II-positive TEC (315). By contrast, other "classic" pancreatic hormones, such as glucagon and somatostatin, appear to be expressed intrathymically by macrophages (313). It is noteworthy that in the NOD mouse (that spontaneously develops autoimmune insulin-dependent diabetes), there is a decrease in the expression of the proinsulin genes, as compared with normal mouse strains, whereas no changes were observed in glucagon or somatostatin (316). This finding raised the hypothesis that decreased intrathymic expression of insulin might favor the escape of thymocytes potentially able to recognize insulin in the periphery.

D. Neuropeptide expression by the thymic microenvironment
One of the first demonstrations of intrathymic production of neuropeptides was the observation of vasopressin and oxytocin, as well as of their corresponding mRNA transcripts, in the human thymus (317). Further work established the thymic epithelium (including TNCs) as a source of these neurohormones both in vivo and in vitro (318, 319, 320). Moreover, in the thymus, the biosynthesis of oxytocin and arginine-vasopressin also results from cleavage of corresponding neurophysins, similar to what happens in the hypothalamus (321). Whether these hormones are actually secreted has not been determined. Recent biochemical and ultrastructural findings suggest that after processing, oxytocin is directly exported to the surface membrane rather than directed to secretory vesicles (322). This finding indicates that, at least in part, fragments of these peptidic hormones are presented to differentiating thymocytes in the context of the MHC molecules expressed on the TEC surface. From a conceptual viewpoint, in addition to being paracrinally secreted, intrathymic expression of peptidic hormones may also be related to negative selection of those thymocytes that could potentially recognize these molecules in the periphery of the immune system, causing antihormone T cell autoreactivity.

In addition to "classical" neurohypophyseal hormones, neuropeptides typically found and secreted in the hypothalamus have been detected in thymic cells. Using ligand binding as well as ELISA, LH-RH was found in human thymocytes (140). It is noteworthy that the control of thymic LH-RH may differ from what occurs in the hypothalamus. As studied in rats, an increase in the LH-RH contents in the thymus after castration was found, and such an increase was prevented by testosterone replacement (323). This contrasted with the decrease in the hypothalamic contents of LH-RH in castrated animals.

The intrathymic production of CRH has also been demonstrated by several experimental approaches (324, 325, 326) and appears to be phylogenetically conserved, as ascertained by immunocytochemistry (327). In one double labeling immunocytochemical study, macrophages were incriminated as the cell source of intrathymic CRH (328). Together with findings showing the production of ACTH and corticosterone by TEC, this raises the hypothesis that a whole circuit, similar to that seen in the hypothalamus-pituitary-adrenal axis, may occur intrathymically. Nevertheless, in terms of biological responses, the existence of such a cascade and how it might be regulated has not been completely demonstrated in the thymus.

In keeping with the expression of the POMC gene is the detection of ß-endorphin in thymus extracts, as ascertained by RIA, as well as in TEC (329, 330, 331). Intrathymic opioid production apparently is not restricted to ß-endorphin, since met-enkephalin and leu-enkephalin have been detected by immunocytochemistry in both lymphoid and microenvironmental compartments of the rat thymus (329, 330, 331, 332), a finding in keeping with evidence for pro-enkephalin A gene expression in the organ (333). Somatostatin gene expression has also been identified in the rat thymus, at the levels of mRNA and translated peptide (334, 335). Recent data suggest that somatostatin is expressed in macrophages and restricted to the medulla (313, 314).

Cloning of a TRH precursor has recently been reported in the rat thymus. Prepro-TRH mRNA was initially defined by RT-PCR using specific nucleotide primers, and the amplified gene product was then sequenced. The TRH peptide itself was further revealed by reverse phase HPLC analysis (266). Unfortunately, this study did not determine which cell type is responsible for intrathymic TRH production. In any case, considering the local production of TRH together with the intrathymic expression of the TRH receptor and the fact that this neuropeptide is able to enhance BrDu uptake by thymocytes (152), it is plausible that an autocrine/paracrine physiological TRH-mediated circuit also exists in the thymus.

Lastly, intrathymic gene expression for VIP should be noted. Various methodological approaches have shown that CD4⁺CD8⁺ double-positive as well as CD4⁺ and CD8⁺ single-positive thymocytes express this neuropeptide (276). This agrees with previous findings from the same research group showing in situ immunoreactivity for VIP in many thymocytes (335). As discussed earlier, the autocrine/paracrine secretion of VIP by thymocytes, together with its role in counteracting apoptosis induced by glucocorticoid hormones, places this neuropeptide as one further player in the general process of generating the T cell repertoire in the thymus (336). This hypothesis is further supported by data indicating that VIP enhances the antigen-induced differentiation of CD4⁺CD8⁺ immature thymocytes to the CD4⁺ simple positive mature stage after interacting with specific antigen presenting cells (337). Similarly, enhancement of VIP production upon mitogenic and antigenic stimulation has been recently shown (338).

The intrathymic expression of various hormones and neuropeptides is summarized in Tables 2 and 4.

	X. Conclusions and Major Questions to Be Addressed

Top Abstract I. Introduction II. The Thymic Microenvironment... III. Neuroendocrine Control of... IV. Thymic Endocrine Function... V. Proliferation of Thymic... VI. Hormonal Modulation of... VII. Is Thymocyte Traffic... VIII. Expression of Receptors... IX. Intrathymic Expression of... X. Conclusions and Major... References

 
The findings discussed herein clearly indicate that the thymus is physiologically under neuroendocrine control. It is apparent that circulating levels of distinct hormones and neuropeptides are necessary to maintain various biological functions related to both microenvironmental and lymphoid cells of the organ (summarized in Fig. 8). However, such neuroendocrine control of the thymus appears to be far more complex, with possible intrathymic biological circuitry involving in situ production of these mediators, as well as the influence of neurotransmitters, which were not targeted herein for discussion, being extensively reviewed elsewhere (3, 339).

View larger version (44K): [in this window] [in a new window]   Figure 8. Pleiotropic nature of the neuroendocrine control of thymus physiology. In this schematic representation, neuroendocrine stimuli are provided by the hypothalamus-pituitary axis, thyroid gland, or opioid-producing cells. Hormones and neuropeptides can act directly on thymocytes, modulating their proliferation rate. Additionally, intrathymic T cell maturation can be indirectly controlled via changes in the behavior of the thymic microenvironment, exemplified herein by TEC. Accordingly, thymic hormone production and expression of ECM ligands and receptors can be hormonally regulated, with consequent effects on proliferation, differentiation, and migration of thymocytes.

In conjunction with this point, another still unresolved question is how hormone-mediated paracrine circuits are regulated.

Since one major function of the thymus is to generate a T cell repertoire, simultaneously bearing diversity but not autoreactivity, it is crucial to have precise knowledge of the extent to which such a process is under neuroendocrine control. Phenotypic analysis of the Vß and V gene rearrangements in thymocytes derived from fetal thymus organ cultures subjected to distinct hormones or neuropeptides will be of interest. We anticipate that the use of different models of knockout mice as well as animals in which hormone-specific transgenes are coupled to thymus-specific promoters may be useful in further determining the relative role of each hormone or neuropeptide in the general process of shaping the T cell repertoire. In this context, it is worthwhile recalling that conflicting results are likely to occur when a role for a given hormone is approached in mice in which the corresponding gene (or the gene for the corresponding receptor) has been inactivated. This is apparently the case of PRL or PRL receptor knockout mice, which apparently develop an efficient immune system, with a normal thymocyte differentiation profile (177, 178). This clearly indicates that PRL/PRL receptor-mediated interaction is not crucial for thymus development. Yet, such data should be viewed with caution, as these mice never had functional PRL or PRL receptor. As in several other redundant systems, it is possible that further biological pathway(s) replace(s) the one triggered by PRL-mediated interactions. Perhaps a better model would be a genetically engineered animal in which the receptor can be activated or de-activated by gene manipulation in adult life. In chickens deprived of major neuroendocrine centers by surgical removal of embryonic prosencephalon, thymocyte development ceases with accumulation of immature CD4^-CD8^- thymocytes within the organ (340). This picture is strikingly reversed by embryonic pituitary gland engraftment or by supplying recombinant PRL, thus emphasizing that PRL may be active, particularly in relation to thymocyte differentiation.

In a second vein, several aspects of thymus physiology have not yet been studied in PRL receptor knockout animals. One example is cell traffic, e.g., thymocyte traffic. We have recently noted that, in spite of a normal CD4/CD8-defined differentiation pattern, the expression of fibronectin receptors by thymocytes from GH receptor null animals is altered compared with the corresponding age-matched wild-type mice, suggesting that T cell migration may be altered in these mice (our unpublished data).

A third aspect to be recalled is the paucity of information regarding the direct effects of hormones upon the thymus in humans. Although thymulin production is definitively up-regulated by several hormones including T₄, PRL, and GH (76, 84), the various aspects of intrathymic T cell differentiation in humans have not been directly assessed (reviewed in Ref. 341). Unfortunately, in one study on long-term treatment of monkeys with GH, the thymus was not analyzed (342).

If we consider that abnormal generation and/or expansion of autoreactive T cells from the thymus can be influenced by the neuroendocrine system, it will be important to develop therapeutic strategies based on the neuroendocrine-mediated manipulation of T cells when they are undergoing intrathymic differentiation.

	Acknowledgments

 
The authors thank Dr. F. Homo-Delarche for helpful discussion, Mrs. Martine Netter and Heloisa Nogueira Diniz for computer drawings, Mrs Catherine Slama for typing the manuscript, and Mrs. D. Broneer for English review.

	Footnotes

 
Address reprint requests to: Wilson Savino, Laboratory on Thymus Research, Department of Immunology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Avenue Brasil 4365, Manguinhos 21045-000, Rio de Janiero, Brazil. E-mail: savino{at}gene.dbbm.fiocruz.br

¹ This work was partially supported by grants from Brazilian Research Council (CNPq, Brazil) and Program for Excellency in Science (PRONEX/CNPq, Brazil), Centre National de la Recherche Scientifique (CNRS, France), Institut National de la Santé et de la Recherche Médicale (INSERM, France), Oswaldo Cruz Foundation (FIOCRUZ, Brazil) and Programme International de Cooperation Scientifique (PICS, France-Brazil).

	References

Top Abstract I. Introduction II. The Thymic Microenvironment... III. Neuroendocrine Control of... IV. Thymic Endocrine Function... V. Proliferation of Thymic... VI. Hormonal Modulation of... VII. Is Thymocyte Traffic... VIII. Expression of Receptors... IX. Intrathymic Expression of... X. Conclusions and Major... References

 

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