Abstract
Adrenomedullin (AM) is a multifunctional peptide endowed with various biological actions mediated by the interaction with the calcitonin receptor-like receptor (CLR), which couples to the receptor activity-modifying proteins 2 or 3 (RAMP2 or RAMP3) to form the functional plasma membrane receptors AM1 and AM2, respectively. In this study, we investigated for the first time the expression and localization of AM, CLR, RAMP2 and RAMP3 in human thymic tissue from newborns and in primary cultures of thymic epithelial cells (TECs) and thymocytes. Immunohistochemical analysis of thymic tissue showed that both AM and RAMP2 are abundantly expressed in the epithelial cells of medulla and cortex, blood vessels and mastocytes. In contrast, RAMP3 could not be detected. In cultured TECs, double immunofluorescence coupled to confocal microscopy revealed that AM is present in the cytoplasmic compartment, whereas RAMP2 could be detected in the cytoplasm and nucleus, but not in the cell membrane. At variance with RAMP2, CLR was not only present in the nucleus and cytoplasm of TECs, but could also be detected in the cell membrane. The nuclear and cytoplasmic localizations of RAMP2 and CLR and the absence of RAMP2 in the cell membrane were confirmed by western-blot analysis performed on cell fractions. AM, RAMP2 and CLR could also be detected in thymocytes by means of double immunofluorescence coupled to confocal microscopy, although these proteins were not present in the whole thymocyte population. In these cells, AM and RAMP2 were detected in the cytoplasm, whereas CLR could be observed in the cytoplasm and the plasma membrane. In conclusion, our results show that the AM system is widely expressed in human thymus from newborns and suggest that both AM1 receptor components CLR and RAMP2 are not associated with the plasma membrane of TECs and thymocytes but are located intracellularly, notably in the nucleus.
Highlights
The thymus provides a variety of specialized microenvironments that support the production of self-tolerant T cells starting from immature precursors [1]
In our previous studies we observed that AM, RAMP2 and RAMP3 are expressed in rat thymus and investigated the role for AM and its AM1 and AM2 receptors in the control of thymic functions
The present study demonstrates for the first time that AM and RAMP2, but not RAMP3, are largely distributed in the newborn human thymus, where they can be found in blood vessels, mast cells and the epithelial compartments of both the cortex and the medulla
Summary
The thymus provides a variety of specialized microenvironments that support the production of self-tolerant T cells starting from immature precursors [1]. As reviewed recently [2], each maturation event of T cell takes place in a discrete region of the thymus and relies on the interaction of thymocytes with specialized thymic epithelial cells (TECs), located in both the cortex (cortical thymic epithelial cells, cTECs) and the medulla (medullary thymic epithelial cells, mTECs). T cell progenitors enter the thymus at the cortex/medulla border via post–capillary venules and migrate toward the capsule in response to chemokine signalling. Thymocytes undergo positive selection by cTECs and migrate to the medulla where they are screened for reactivity to tissue-restricted self antigens expressed by mTECs [3]. Developing thymocytes and TECs establish a mutual ‘‘cross talk’’ that is necessary for the functional maturation of both types of cells [2]. Thymic functions are regulated by various peptides, such as ghrelin [5], leptin [6,7], neuronal growth factor [8,9] and interleukins [10]
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