Abstract
BackgroundThe thymic epithelium is the major microenvironmental component of the thymus, the primary lymphoid organ responsible for the generation of T lymphocytes. Thymic epithelial cells (TEC) control intrathymic T cell differentiation by means of distinct types of interactions. TEC constitutively produce chemokines and extracellular matrix ligands (such as laminin and fibronectin) and express corresponding receptors, which allow thymocytes to migrate in a very ordered fashion. We previously showed that laminin mediates TEC/thymocyte interactions in both mice and humans. More recently, we used RNAi technology to knock-down the ITGA5 gene (which encodes CD49e, the integrin α-chain subunit of the fibronectin receptor VLA-5) in cultured human TEC. Using a similar strategy, herein we knocked-down the ITGA6 gene, which encodes CD49f, the α-chain of two integrin-type laminin receptors, namely VLA-6 (α6β1) and α6β4.ResultsWe first confirmed that RNAi-induced knock-down of the ITGA6 gene was successful, at both transcription and translational levels, with a significant decrease in the membrane expression of CD49f, apart from CD49b, CD49c and CD49d, ascertained by cytofluorometry on living TEC. We also demonstrated that such knock-down promotes a decrease in cell adhesion to laminin. Using quantitative PCR, we demonstrated that gene expression of other integrin α-chains were concomitantly down-regulated, particularly those which form other laminin receptors, including ITGA1, ITGA2 and ITGA7. Interestingly enough, LAMA1 gene expression (whose corresponding protein chain is part of laminin-111) was largely increased in ITGA6 knocked-down TEC cultures. Lastly, the network complexity of gene expression under ITGA6 influence is much broader, since we found that other cell migration-related genes, namely those coding for various chemokines, are also modulated when IGTA6 is knocked-down.ConclusionThe data presented herein clearly show that down regulation of ITGA6 gene in the human thymic epithelium triggers a complex cascade of effects upon the expression levels of several other cell migration-related genes, including extracellular matrix and chemokine ligands and receptors. Taken together, these data unravel the concept that the expression of genes involved in controlling of thymocyte migration by the thymic microenvironment should be regarded as complex networks, so that a defect in the expression of one single gene may reflect in an amplified cascade with functional consequences for TEC adhesion onto the natural ligand and potential consequences upon the normal patterns of TEC/thymocyte interactions.
Highlights
The thymic epithelium is the major microenvironmental component of the thymus, the primary lymphoid organ responsible for the generation of T lymphocytes
We demonstrated in both mice and humans that developing thymocytes and Thymic epithelial cells (TEC) express constitutively the laminin receptor VLA-6, which participates in heterocellular cell adhesion and migration events, which could be significantly disrupted by using anti-VLA-6 antibody applied to growing TEC prior to co-culturing with thymocytes [8,9,10]
We found that several other cell migration-related genes were modulated secondary to treating human TEC with ITGA6 specific siRNA, pointing to the notion that intra-TEC expression of genes involved in controlling thymocyte migration by the thymic microenvironment should be regarded as complex networks
Summary
The thymic epithelium is the major microenvironmental component of the thymus, the primary lymphoid organ responsible for the generation of T lymphocytes. It has been largely demonstrated that, for such a process to occur normally, it is crucial that developing thymocytes interact to nonlymphoid cells of the organ, the thymic microenvironment [1] This tridimensional network is composed of a variety of cell types; the thymic epithelial cells (TEC) being the most conspicuous elements, nonepithelial dendritic cells, macrophages and, to a lesser extent fibroblasts, play a role in the general process of intrathymic T cell differentiation. Supramolecular ECM arrangements may function as a conveyor belt, allowing an ordered migration of thymocytes within the organ [5] In this context, we have demonstrated that interactions mediated by fibronectin and its receptor VLA-5 (the integrin a5b1 or CD49e/ CD29) influence TEC thymocyte adhesion, as demonstrated by the use of neutralizing anti-CD49e monoclonal antibodies [6] or RNA interference to abrogate translation of the CD49e protein [7]. We demonstrated in both mice and humans that developing thymocytes and TEC express constitutively the laminin receptor VLA-6 (the integrin a6b1 or CD49f/CD29), which participates in heterocellular cell adhesion and migration events, which could be significantly disrupted by using anti-VLA-6 antibody applied to growing TEC prior to co-culturing with thymocytes [8,9,10]
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