TECHNICAL NOTE |
Correspondence to: Georg Wick, Inst for General and Experimental Pathology, U. of Innsbruck, Fritz-Pregl-Str. 3/IV, Innsbruck A-6020, Austria.
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Summary |
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Thymic nurse cells (TNC) and T-cell stromal rosettes (ROS) are two in vivo models for stromal cell-thymocyte interactions. We describe a simplified enrichment method for TNC and ROS that overcomes the necessity for large amounts of tissue. The complexes were further analyzed with confocal microscopy, and three subunits of ROS were defined on the basis of their central cell phenotype, i.e., macrophage, dendritic, or epithelial cell rosettes. Because adhesion molecules are proposed to play a crucial role in T-cell development, we investigated CD44, LFA-1, and ICAM-1 expression in such complexes. The epithelial component of TNC expresses CD44 and ICAM-1, whereas intra-TNC thymocytes are LFA-negative. With regard to ROS, all subsets expressed CD44, and macrophage and dendritic cell ROS were also ICAM-1-positive and LFA-1-positive. The current protocol opens the possibility for further in vivo analysis of stromal cell-thymocyte interactions, e.g., for studies of scarce gene mutant mice. (J Histochem Cytochem 45:1293-1297, 1997)
Key Words: thymic nurse cell, thymic rosette, thymocyte, confocal microscopy
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Introduction |
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Intrathymic T-cell development is characterized by a corticomedullary migration during which thymocytes undergo maturation and finally emigrate to the periphery (
Two in vivo models for such stromal cell-thymocyte interactions are the thymic nurse cells (TNC) and thymocyte stromal cell rosettes (ROS). TNC are cortical multicellular complexes composed of intra-TNC thymocytes (TNC-T) enclosed within epithelial cell vacuoles (
During their intrathymic journey, thymocytes rely on adhesion molecules and receptors for extracellular matrix proteins as important driving force (
A main limitation of current enrichment methods for studying such thymic cellular complexes is the large amounts of tissue needed. In this report we describe a simplified enrichment method for TNC and ROS that allows experiments with small amounts of tissue (e.g., two thymuses). In addition to quantification, the phenotype of the heterotypic cellular complexes can be studied. Therefore, using confocal scanning microscopy, we divided ROS into three subsets on the basis of their central cell phenotypes: macro-phages (M-ROS), dendritic (DC-ROS), or epithelial cell (Ep-ROS) rosettes. Furthermore, TNC and ROS expression of CD44, LFA-1, and ICAM-1 adhesion molecules was investigated.
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Materials and Methods |
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Mice
Four- to 6-week-old female C57BL/6 mice were obtained from the Central Laboratory Animal Facility, University of Innsbruck, Medical School. They were maintained at a 12-hr light-dark cycle with food and water ad libitum.
Enrichment of TNC and ROS
Using a modification of a method previously described by
Immunostaining
For phenotyping of TNC and ROS, we used double staining immunofluorescence. Primary and secondary antibodies were diluted in PBS with 1% bovine serum albumin and 10% normal mouse serum, and each labeling step lasted 30 min at room temperature (RT). The TNC epithelial component (Ep-TNC) was identified with rabbit polyclonal anti-human pan-keratin (Dako; Glostrup, Denmark), and TNC and TNC-T nuclei were stained with propidium iodide. ROS central cells were labeled with the rat monoclonal antibody (MAb) F4/80 anti-M (
To quantify ROS subsets, we used an immunocytochemical double staining technique. Primary and secondary antibodies were diluted in buffer (Tris 0.01 M, NaCl 0.15 M) containing 1% bovine serum albumin and 10% normal mouse serum. Each sample was preincubated in an NaN3-H2O2 solution for 30 min to block endogenous peroxidase activity, and each labeling step lasted for 30 min at RT. As secondary antibodies, we used horseradish peroxidase-conjugated rabbit anti-rat Ig (Dako), alkaline phosphatase-conjugated F(ab')2 fragment donkey anti-rabbit IgG, biotin-conjugated F(ab')2 fragment rabbit anti-hamster IgG (Jackson Immunoresearch), and alkaline phosphatase-conjugated streptavidin (Boehringer Mannheim). For detection, Fast DAB with metal enhancer and Fast Red enzyme substrate were used, respectively (Sigma; St Louis, MO). In all immunostaining experiments, nonimmune species-matched Ig or serum was used as negative control for each primary antibody. Optimal dilution of all primary antibodies and visualization reagents was determined in pilot studies.
Microscopic Analysis
Immunoenzymatic staining of ROS was analyzed by light microscopy. Immunofluorescence-stained samples were analyzed with an LSM 10 confocal laser scanning microscope (Zeiss; Oberkochen, Germany). Digital images of fluorescence were acquired in response to excitation with a helium neon laser (543 nm), filter setting BP 575-640, for cells stained with TRITC or Cy-Chrome, and an argon laser (488 nm), filter setting BP 530/30 or LP 520, for FITC-stained cells. Images were collected at a scan rate of 8 sec. Digital images were transferred to a high-resolution RGB color video photomonitor (Lucius and Baer; Geretsried, Germany) and were photographed with Fujicolor Super HG 200 film.
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Results and Discussion |
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This simplified enrichment method, modified from a protocol described by
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Phenotype studies of multicellular complexes require multiple markers and good morphological definition. That is especially true for TNC with their three-dimensional structure and up to 200 thymocytes enclosed within individual vacuoles (Figure 2a). Therefore, we analyzed TNC and ROS obtained with this simplified method using confocal laser scanning microscopy. The Ep-TNC can be identified by its cyto-keratin expression, which builds up a cytoplasmic network (Figure 2b). Furthermore, TNC-T can have their nuclei visualized by DNA markers, like propidium iodide (Figure 2c), or can be identified by membrane markers, e.g., CD3 (Figure 2d). With regard to adhesion molecule expression, Ep-TNC are positive for CD44 (Figure 2e). Interestingly, the large majority of TNC express ICAM-1 (Figure 2f), whereas TNC-T appear to be LFA-1-negative. Thymocytes are mainly LFA-1-positive, and it has been shown that LFA-1/ICAM-1 interaction mediates thymocyte-thymic epithelial cell binding in vitro (
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ROS were grouped into three subsets on the basis of their stromal components. A quantitative analysis using immunocytochemically stained samples showed a majority of M-ROS (63%), together with 27% of DC-ROS and 10% of Ep-ROS (Figure 3). All ROS subsets show CD44-positive stromal cells (Figure 2g-i), and M
- and DC-ROS also express LFA-1 and ICAM1 (data not shown), suggesting that these adhesion molecule can be involved in the formation of such complexes.
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In conclusion, this simplified method for TNC and ROS enrichment, combined with subtyping of ROS complexes, will allow further investigation into the role of such complexes in T-cell development. Our group is currently addressing the question of when TNC and ROS are formed during T-cell development and which adhesion molecules are involved, using a gene-targeted mouse with different blocks in T-cell differentiation and adhesion molecule expression.
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Acknowledgments |
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Supported by the Austrian Research Council (grant no. 10654) and by the EU Human Capital Mobility Program (contract no. CHRXCT 930246) (to GW).
Received for publication February 13, 1997; accepted April 24, 1997.
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