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First published online 14 March 2006
doi: 10.1242/jcs.02840

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Characterization of a conduit system containing laminin-5 in the human thymus: a potential transport system for small molecules

¹ Section for Transplantation Immunology and Immunohematology, Center for Medical Research, University of Tübingen, 72072 Tübingen, Germany
² Department of Nephrology and Rheumatology, University Hospital Göttingen, 37075 Göttingen, Germany
³ Institute for Physiological Chemistry, Münster University Hospital, 48149 Münster, Germany
⁴ Department of Neurology, University Hospital Tübingen, 72076 Tübingen, Germany
⁵ Center for Biochemistry, Department of Dermatology, and Center for Molecular Medicine, University of Cologne, 50931 Cologne, Germany
⁶ Department of Anatomy and Cell Biology and Faculty of Dentistry, McGill University, Montreal, Quebec H3A 2B2, Canada
⁷ Max Planck Institute of Biochemistry, Department of Molecular Medicine, 82152 Martinsried, Germany
⁸ Center for Molecular Biology of Plants, University of Tübingen, 72076 Tübingen, Germany
⁹ Max-Planck-Institute for Developmental Biology, 72076 Tübingen, Germany

View larger version (100K): [in a new window]   Fig. 1. Molecular characterization of medullary thymic conduits. The micrographs show single (A-D) or double (E-N) immunofluorescence staining with Cy3- or FITC-labeled antibodies counterstained in blue with DAPI to visualize cell nuclei. (A,B) Labeling with antibody specific for the 2 chain of the LN-5 isoform showed an exclusive staining of structures located in the medulla (A). Upon enlargement, bi-membranous structures or conduits, with an average diameter of 2 µm, can be clearly detected (B). (C,D) The laminin 3 chain (LN3; C) and the laminin ß3 chain (LN5ß3; D), both components of the LN-5 isoform, are also detected in the conduits. (E-H) Typical basement membrane components are found in LN-5-containing conduits, as shown by double immunofluorescence staining with antibody specific for the ß3 chain (LN5ß3; G) or the 2 chain (LN52; E,F,H) of the LN-5 isoform plus collagen type IV (E), perlecan (F), nidogen (G) and EHS-laminin (EHS-LN; H) antisera. (I-K) Fibrillins and tenascin-C are also associated with the conduits. This is shown by double labeling with fibrillin-1 and laminin 2 chain antibodies (I), with fibrillin-2 and laminin ß3 chain antibodies (J) and tenascin-C and collagen type XII antibodies (K). Other collagen types associated with the conduits are the fibrillar collagen type I (L) and type III (N) or the microfibrillar collagen type VI (M). Here, the conduits were labeled with laminin 2 chain (L), laminin ß3 chain (M) or EHS-laminin (N) antibodies. Bar, 10 µm.

View larger version (54K): [in a new window]   Fig. 2. 3D reconstruction of the conduits. A thymic cryostat section 10 µm in thickness was labeled with antibodies against the laminin 2 chain (shown in red) and tenascin-C (shown in green) and subjected to confocal microscopy. An automated `Z-scan' generated a set of merged 2D pictures that were fused into a 3D depiction of the area of interest by the Imaris® software. The 3D pictures can be sectioned at every angle. In the longitudinal section (A), as well as in the cross-section (B), a tubular structure consisting of a LN-5-containing membrane (red) surrounded by a tenascin-C-containing sheath can be clearly observed. [A virtual journey through the thymic conduits can be viewed at http://www.wewe-design.de/klein.]

View larger version (126K): [in a new window]   Fig. 3. Electron microscopic analysis of the conduits. (A) Ultrathin thymic cryosections were labeled with antibodies against the laminin 2 chain, which were detected by nanogold-coupled secondary antibodies. After silver enhancement, labeling of a membrane surrounding an area rich in fibrillar collagens could be observed. (B) Transmission electron microscopy of the thymic medulla revealed densely packed collagen bundles surrounded by a basement membrane. These conduit-like structures are enwrapped by a cell that is rich in intermediate filaments. Note the desmosome at the top of the right conduit. Bars, 1 µm.

View larger version (75K): [in a new window]   Fig. 4. Connections of conduits to blood vessels and Hassal's bodies. (A-C) Double immunofluorescence staining of a thymic cryostat section with antibody against the laminin 2 chain (A) and the antiserum against fibrillin-1 (B) revealed a colocalization of both antigens in the conduits; by contrast, only fibrillin-1 can be detected in the blood vessels. In the merged picture (C), a nuclear DAPI staining is included. (D-F) Conduits are also connected to Hassal's bodies as shown in the double immunofluorescence staining using antibody against the laminin 2 chain (D) and an antiserum against ß-catenin (E). In the overlay picture (F), which also includes the nuclear blue staining, the direct association of the conduits with the Hassal's body can be observed. Bar, 10 µm.

View larger version (101K): [in a new window]   Fig. 5. Associations of conduits with non-lymphoid cells of the thymic medulla. (A-C) The micrographs show double immunofluorescence staining of a thymic cryostat section with antibodies against (C) cytokeratin, which detects TECs, and antibodies against (B) laminin 2 chain, which reveals thymic conduits. The merged picture (A), which is counterstained with DAPI to visualize the cell nuclei, revealed that the TECs are closely associated with the conduits. (D-F) Double immunofluorescence staining with antibodies against (F) CD11c, which detects dendritic cells, and antibodies against (E) laminin ß3 chain, which reveals thymic conduits, especially in the merged picture that is counterstained with DAPI (D), reveals that CD11c+ dendritic cells can be detected in close vicinity to the thymic conduits. (G-I) TECs express MHC class II molecules on their cell surfaces as indicated by the double immunofluorescence staining with antibodies against cytokeratin (I) and antibodies against HLA-DR class II molecules (H). The overlay picture clearly shows the colocalization (G). Bar, 10 µm.

View larger version (72K): [in a new window]   Fig. 6. Synthesis of LN-5 by TECs. (A) RT-PCR analysis of isolated primary TECs (PTEC) and CD11c+ dendritic cells (CD11c+) showed that only primary TECs synthesize the 3, ß3 and 2 chains of LN-5. Total RNA was extracted from both cell types and from the lung adenocarcinoma cell line A549, which served as a positive control. After reverse transcription, cDNA quality was checked by ß-actin PCR. Equal amounts of cDNA were subjected to PCR analysis for the laminin 3, ß3 and 2 chains. Whereas the control cell line A549 and primary TECs synthesize all three chains of LN-5, dendritic cells do not. (B) By immunoprecipitation of TEC lysates with antibodies against the laminin 3 and 2 chain, and subsequent immunoblotting (WB) of the immunoprecipitates (IP) with the anti-laminin 2 chain (LN-2-chain) antibody, the processed form of laminin 2 chain at 105 kDa could be detected. Co-precipitation of the laminin 3 chain with laminin 2 revealed an intact LN-5 isoform. The bands at 55 kDa represent the heavy chains of the precipitating antibodies. (C,D) Immunofluorescence staining of TECs cultured in vitro with the laminin 2 chain antibody showed a strong staining signal in red, the cell nuclei are counterstained with DAPI in blue (D). Dendritic cells, however, did not synthesize LN-5 since no labeling of the dendritic cells in vitro could be detected with the laminin ß3 chain antibody, only the DAPI counterstaining of the nuclei is visible (C). Bar, 15 µm.

View larger version (76K): [in a new window]   Fig. 7. Cell attachment of dendritic cells (DCs) and TECs to different ECM molecules. Isolated DCs and TECs were allowed to adhere to different plastic-immobilized ECM molecules including LN-5 (A,C,D), LN-10/11 (B,E,F), tenascin-C (G,H), the N-terminal half of fibrillin-1 (I) and the C-terminal half of fibrillin-1 (J-L). 1 µl of each protein was spotted onto Petri-dishes and allowed to air dry. After 1 hour of incubation of the two cell types with the immobilized proteins, the unbound cells were washed away. DCs attached strongly to the area coated with LN-10/11 (B), whereas only a moderate binding to LN-5 was detectable (A). Primary TECs attached equally well to LN-5 (C), LN-10/11 (E), tenascin-C (G) and to the C-terminal (C-term.) half of fibrillin-1 (J), but not to the N-terminal (N-term.) half of fibrillin-1 (I). Cell attachment to all the adhesive ECM molecules was concentration dependent. A representative example is shown for fibrillin-1 (J-L). Cell attachment to fibrillin-1 could only be observed using concentrations >0.05 µg/µl (K,L). Interaction of TECs with LN-5, LN-10/11 and tenascin-C could be completely blocked by pre-incubation of the cells with a function-blocking ß1-integrin antibody (D,F,H). In A and B, the dotted lines delineate the border between the laminin-coated areas and the uncoated areas. Note that outside the laminin-coated areas, only weak background binding is still observed after washing. In all the other micrographs (C-L), the ECM-coated area is located in the center of the pictures in a circular area. In the uncoated area outside the circles, almost no cell attachment could be detected. Bars, 300 µm.

View larger version (117K): [in a new window]   Fig. 8. Passive transport of small molecules through the thymic medullary conduits. Thymic fragments were incubated with 10 kDa FITC-dextran (A,C,D1) or 500 kDa FITC-dextran (B) for 30 minutes (A,C,D) or 2 hours (B) at 4°C (A,B) or 37°C (C,D). After fixation, cryostat sections of the incubated fragments were labeled with antibodies against the laminin 2 chain (A,B,D2) or CD31 (C) followed by Cy3-labeled secondary antibody. All sections were also labeled with DAPI (blue staining) to identify the cell nuclei. (A) After 30 minutes at 4°C, the 10 kDa FITC-dextran can be detected in blood vessels and in conduits (identified by the LN-5 staining), whereas the 500 kDa FITC-dextran cannot be found in conduits (red staining) even after two hours of incubation (B). After 30 minutes at 37°C, the 10 kDa FITC-dextran is still restricted to blood vessels and to conduits. This is shown in (C) by a labeling of blood vessels with the CD31 antibody and in D1 by LN-5 labeling. D2, FITC-dextran; D3, merged image with DAPI staining. Note that there is no FITC-labeling visible outside the conduit system (C,D).

View larger version (69K): [in a new window]   Fig. 9. Schematic representation of the architecture of the thymic conduits. The central collagen core of fibrillar collagens is surrounded by a continuous basement membrane (red). The layer shown in green is a microfibrillar layer containing fibrillins and tenascin-C. The conduits are enwrapped by TECs. The nucleus is shown in blue.