Thymic vasculature: organizer of the medullary epithelial compartment?

Monica Anderson1, Susan K. Anderson2 and Andrew G. Farr1,3

1 Departments of Biological Structure,
2 Medicine, and
3 Immunology, School of Medicine, University of Washington, Seattle, WA 98195, USA

Correspondence to: A. G. Farr, Department of Biological Structure, School of Medicine, University of Washington, Box 357420, Seattle, WA 98195-7420, USA


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Immunohistochemistry
 Results and discussion
 References
 
The epithelial component of the thymic environment is organized into discrete cortical and medullary compartments that mediate different aspects of thymocyte differentiation. The processes controlling the growth and organization of these epithelial compartments are poorly defined. In this study we have used a novel approach to define the three-dimensional organization of thymic epithelial (TE) compartments to demonstrate that the organization of the medullary TE compartment is very complex. A spatial relationship of medullary thymic epithelium with vascular elements of the thymus was demonstrated by simultaneous immunohistochemical labeling of vascular elements and medullary TE. Medullary TE was often arranged as perivascular cuffs surrounding intermediate-sized vessels, but was not associated with either the capillary network or large centrally located vessels. Similar analyses of RAG-2–/– thymi revealed a striking physical association of medullary TE with vascular elements. Ultrastructural analysis of the RAG-2–/– thymus indicated a preferential association of focal accumulations of medullary TE with post-capillary venules. These data suggest that discrete segments of the thymic vasculature provide cues that act in concert with thymocyte-derived stimuli to effect normal development of the thymic environment.

Keywords: cellular differentiation, endothelial cells, stromal cells, thymus


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Immunohistochemistry
 Results and discussion
 References
 
Shaping of the TCR {alpha}ß T cell repertoire involves several distinct compartments of the thymic environment. The cortical compartment, composed largely of a reticular network of epithelial cells that present an array of peptide–MHC complexes, plays a critical role in the positive selection process. Negative selection of thymocytes bearing high-affinity receptors for self-peptide–MHC complexes and further differentiation of CD4/CD8 single-positive thymocytes can occur within the medullary/cortico-medullary compartments of the thymus (discussed in 1).

The cellular composition of the medulla is heterogeneous, consisting of bone marrow-derived and epithelial cells (2). Medullary thymic epithelium (TE), which can be distinguished from cortical TE by morphologic and phenotypic criteria (3), are themselves heterogeneous (4). The medullary TE elements express a wide range of peptide hormones normally associated with other endocrine tissues (5) and elaborate chemokines thought to contribute to the organization of the thymic environment (6). Some medullary TE express high levels of several co-stimulatory molecules implicated in antigen presentation/T cell activation, including CD40 (7) and B7-1 (8), and have been shown to process and present protein antigens to T cells in vitro (9). The tolerogenic capacity of medullary TE has been demonstrated in murine transgenic models where expression of the potential tolerogen was targeted to medullary epithelium (10,11).

It is increasingly apparent that these thymocyte–stromal cell interactions are bidirectional and also play an important role in the development and maintenance of normal TE organization. Disruption of cortical TE development in transgenic mice with an early arrest of thymocyte development points to a role of immigrant T cell progenitors in the developmental program of cortical TE (12). Perturbation of later TCR-dependent stages of thymocyte maturation dramatically interferes with the normal organization of the thymic medullary compartment and the normal differentiation of medullary TE cells, as defined by phenotypic analysis (13). More recently, we have documented significant disruption of the normal compartmentalization of cortical and medullary epithelial elements in thymi from TCR transgenic mice, indicating that alterations in the avidity of TCR-mediated interactions between thymocytes and thymic stromal elements also play a role in the organization of the thymic environment (14).

During the course of this latter study, it became apparent that our current understanding of medullary organization was inadequate. We report here, based on three-dimensional reconstruction of the immunohistochemically defined medulla, that the organization of the normal medullary epithelial compartment is unexpectedly complex with extensive arborization. A consistent spatial relationship between medium-sized blood vessels and medullary epithelium was noted in normal thymus tissue. Similar analyses of RAG-2–/– thymic tissue, which lacks the CD3+ thymocyte-derived stimulus for medullary TE expansion/organization, revealed a hypoplastic, but well-organized medullary epithelial compartment that displayed a remarkably close spatial association with blood vessels. Based on ultrastructural studies, it appears that focal accumulations of epithelial cells preferentially associate with post-capillary venules in the RAG-2–/– thymus. These results suggest that vascular elements may play a key role in the organization and function of the medullary epithelial compartment.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Immunohistochemistry
 Results and discussion
 References
 
Mice
C57B/6 mice and RAG-2-deficient mice were purchased form Jackson Laboratory (Bar Harbor, ME). All protocols involving mice have been reviewed by Department of Comparative Medicine at the University of Washington.

Reagents
Primary antibodies used for immunohistochemical analyses included MECA-32 (15) from the Developmental Biology Hybridoma Bank (Iowa City, IA), 3G10, a rat mAb reactive with medullary TE and raised in this laboratory, and two other mAb that selectively react with medullary TE, ER-TR5 (3) and MTS-10 (16). The 3G10 antibody detects an intracellular constituent of medullary TE cell lines that is resistant to detergent/high-salt extraction and that has an electrophoretic mobility pattern similar to keratin 14 in two-dimensional gels (Farr, unpublished observations). MECA-32 antibodies were modified with NHS-digoxigenin according to the manufacturer. Peroxidase-conjugated goat anti-rat µ chain antibodies were purchased from Pierce (Rockford, IL) and peroxidaseconjugated Fab fragments of goat anti-digoxigenin antibodies were purchased from Boehringer Mannheim (Indianapolis, IN)


    Immunohistochemistry
 Top
 Abstract
 Introduction
 Methods
 Immunohistochemistry
 Results and discussion
 References
 
Protocols for light microscopic immunohistochemistry have been previously described in detail (4). Endogenous peroxidase activity was inhibited prior to two-color immunoenzyme labeling. Slides bearing acetone-fixed tissue sections were incubated in PBS containing 1 mM sodium azide, 10 mM glucose and glucose oxidase (0.5 U/ml; Sigma, St Louis, MO) for 30 min at 37°C. Slides were rinsed in PBS prior to immunolabeling. For two-color immunoperoxidase labeling, a mixture of digoxigenin-modified MECA-32 and unconjugated 3G10 was applied as the first-step reagent, followed by peroxidase-conjugated anti-digoxigenin antibodies. After the final rinse in PBS, the slides were incubated in 0.1 M PBS containing 3.3 mM CoCl2, 0.05% 3,3'-diaminobenzidine and 0.033% (v/v) of 30% H2O2. To demonstrate the binding of 3G10, the sections were rinsed again in PBS, and then incubated for 45 min with a peroxidase-conjugated anti-rat µ chain antibody diluted in PBS containing BSA (10 mg/ml) and 10% (v/v) normal mouse serum. The substrate used to demonstrate binding of the 3G10 antibody consisted of 0.1 M sodium acetate containing 0.067% v/v) of 30% H2O2 and 0.01% w/v 3-amino-9-ethylcarbazole (Sigma; stock solution =100 mg/ml in DMSO). After rinsing in PBS, slides were incubated in this mixture for 15 min at room temperature and then rinsed in distilled water. Coverslips were mounted without dehydration, using Fluromount G (Southern Biotechnology, Birmingham, AL).

Image analysis
Entire thymus lobes were serially sectioned at 10–15 µm and processed to demonstrate reactivity with 3G10 antibody. Images were captured using a CCD-72 camera (DAGE-MTI, Michigan City, IN) and Image software (public domain; http://rsb.info.nih.gov/nih-image/download.html), and were imported into Photoshop (Adobe Systems, San Jose, CA), to generate profiles of the thymus and medullary compartments. This set of files was then processed with an image rendering software program (SURFDRIVER, www.surfdriver.com) to generate a three-dimensional representation of the thymus and thymic medullary compartment.

Electron microscopy
Thymus tissue from RAG-2–/– was perfused with a mixed aldehyde fixative and then processed for conventional electron microscopy, using methods previously described (4).


    Results and discussion
 Top
 Abstract
 Introduction
 Methods
 Immunohistochemistry
 Results and discussion
 References
 
The histologically defined medulla has been generally considered to be the lighter staining central region of the thymus, and consists of a mixture of epithelial cells and dendritic cells. To gain a clearer understanding of the organization of the medullary epithelial compartment, we generated three-dimensional representations of the medullary epithelial compartment by integrating complete sets of two-dimensional images that defined the medullary epithelial compartment immunohistochemically. The thymic labeling pattern with 3G10 (Fig. 1a and bGo) is indistinguishable from that of ER-TR5 (3) or with MTS-10 (16) (data not shown). The medullary compartment appeared as multiple irregular foci in some sections and as large single areas in others.



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Fig. 1. Organization of the medullary epithelium in normal mouse thymus. (a) In addition to several large areas of medullary epithelium, there are several small `islands'. Asterisk indicates a large vessel cut in cross-section. (b) Section of thymus labeled with 3G10 antibody. While there is no 3G10+ epithelium associated with a large vessel indicated by an asterisk, a cuff of medullary TE surrounds the profile of a smaller vessel (arrow). Three-dimensional reconstruction of the medullary compartment in normal thymus tissue. The medullary compartment is indicated in yellow-orange color. Panel (c) is to scale. (d) The same lobe as in (c) with the z-axis increased 3 times. Spatial relationships between medullary TE and vascular elements in the normal thymus. Labeling of vascular elements is blue/black; labeling of medullary TE is red. (e) A cuff of medullary TE surrounds an intermediate-sized vessel (arrow), while larger vessels (asterisks) are devoid of associated medullary TE, as are capillaries. (f) Several areas of medullary TE. The upper right one is investing an intermediate-sized vessel (arrow). No vessel is apparent in the upper center island of medullary TE in this section but the vessel above the medullary TE area passes thorough this region several sections away (data not shown). In the lower region of medullary TE, note the orientation along the side of the large vessel possessing smaller branches (arrow), but not on the side devoid of smaller vascular segments. (g) A region of medullary TE investing an intermediate sized-vessel (arrow). As in (f) medullary TE associates along the side of the larger vessel which possesses smaller branches. (h) The large vessel on the left marked with an asterisk has medullary TE associated on the side with an intermediate-sized vessel branch. The vessel in the center transitions from large to intermediate diameter (the large segment is marked with an asterisk). Only the segment with smaller diameter is associated with medullary TE. Panels (a) and (b) x40; (c) and (d) x8; (e)–(h) x45.

 
A representative image of one reconstructed thymus lobe is shown in Fig. 1Go. Figure 1Go(c) depicts a calibrated and normally scaled three-dimensional reconstruction of the 3G10+ medullary epithelial compartment. Figure 1Go(d) displays the same reconstruction with the z-axis expanded to provide better separation of the medullary elements. These images clearly revealed that the organization of the medullary epithelial compartment was much more complex than previously thought, with extensive branching and aborization. It was also readily apparent that many of the smaller foci of medullary epithelium observed in individual sections were actually continuous with each other elsewhere in the thymus.

When considering mechanisms underlying the complex branching organization of the 3G10+ TE, occasional profiles of blood vessels associated with this TE subset raised the possibility that vascular elements could be involved. The location of larger vessels within the thymic medullary compartment has been appreciated for some time based on conventional histological analyses (17,18), but the relationship of these vessels with defined medullary epithelial compartments has not been examined. Although analysis of individual sections indicated that there was no association of the 3G10+ TE with large vessels (Fig. 1bGo), simultaneous localization of vascular elements with the MECA 32 antibody and 3G10+ medullary epithelial cells revealed that vascular elements were routinely found associated within each medullary epithelial region (Fig. 1e–hGo). Interestingly, medullary epithelium was associated with vessels of intermediate diameter (venules or arterioles), but not with small vessels, representing capillaries, or larger medullary vessels (Fig. 1b and e–hGo), suggesting that the association of medullary TE components with the vasculature may reflect unique properties of distinct vascular segments.

Interpretation of the spatial relationships between vascular elements and the medullary epithelial compartment in normal thymic tissue is complicated by the well-recognized expansion and organization of medullary TE in response to stimuli provided by CD3+ thymocytes or mature T cells (13,19). To examine medullary TE organization in the absence of this influence, we performed similar analyses of RAG-2 –/– thymus tissue, where the hypoplastic and disorganized medullary TE presumably reflects the potential of this tissue compartment to develop in response to endogenous or exogenous cues without those provided by CD3+ thymocytes. As shown in Fig. 2Go(a and b), the 3G10+ medullary compartment in the RAG-2–/– thymus consisted of multiple small foci, some of which appeared to be spatially associated with blood vessels. In other areas, 3G10+ cells were distributed as a thin layer investing blood vessels. The distribution of cells with endogenous peroxidase activity, which is largely associated with eosinophils (20), correlated well with the 3G10+ cells and may reflect the production of chemokine, eotaxin, by medullary TE (6). A representative three-dimensional reconstruction of the thymus and medullary TE compartment in a RAG-2–/– mouse is shown in Fig. 2Go(c and d). Although the medullary compartment defined by 3G10 reactivity was hypoplastic, the residual medullary epithelium remained organized as strands or cords with focal expansions and was not dispersed throughout the thymus tissue. The medullary TE in the RAG-2-deficient thymus, while clearly hypoplastic, retained a high degree of compartmentalization and organization.



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Fig. 2. Medullary TE in the RAG-2–/– thymus. (a) Multiple small foci of medullary TE are evident. There is significant endogenous peroxidase activity (small black dots) which is due to enzymatic activity associated with eosinophils. Note the association of these cells with the medullary TE compartment. (b) Medullary TE associated with a large vessel (marked with asterisks) is a thin investment in some areas, with regions of focal expansion where intermediate-sized vessel branch off. Three-dimensional reconstruction of the 3G10+ medullary compartment of RAG-2–/– thymus tissue. The medullary compartment is indicated in yellow. (c) Side view. (d) Top view. Spatial relationships between medullary TE and vascular elements in the RAG-2–/– thymus. Labeling of vascular elements is blue/black; labeling of medullary TE is red. (e) Location of 3G10+ medullary TE is restricted to the immediate vicinity of intermediate and small blood vessels (arrows point to some of them). (f) An occasional larger collection of medullary TE (asterisk) is also adjacent to vascular elements. (g and h) Two more examples of medullary TE associating with vascular elements in the RAG-2–/– thymus. Panels (a) x40; (b) x50; (c) and (d) x16; (e)–(h) x70.

 
Co-localization studies demonstrated that the spatial relationship between blood vessels and 3G10+ TE was more prominent in the RAG-2–/– thymus, presumably due to the lack of thymocyte-derived stimuli (Fig. 1e–hGo). The occasional larger foci of the 3G10+ medullary TE cells were also contiguous with vascular structures. Sensitivity of the 3G10-reactive epitope to paraformaldehyde fixation precluded ultrastructural analysis of the spatial distribution of the 3G10+ cells. However, ultrastructural analysis of RAG-2–/– thymus tissue revealed occasional small islands of perivascular epithelial cells with distinctive small electron dense vesicles (Fig. 3aGo). The vessels associated with these clusters of epithelial cells were classified as small venules, based on their lumenal diameter, the presence of peri-endothelial adventitial cells and the absence of sub-endothelial smooth muscle cells. For comparison, a typical disposition of epithelial cells associated with a small muscular arteriole is shown in Fig. 3Go(b). Studies to determine if this spatial relationship between medullary TE and post-capillary venules in the more complex normal thymic environment is in progress.



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Fig. 3. Ultrastructure of RAG-2–/– medullary epithelium. (a). A rare cluster of epithelial cells associated with a small venule. Arrow, endothelial cell; double arrow, adventitial cell; arrowheads, junctional complexes between epithelial cells. L, lumen; Ly, lymphocyte; E, Epithelial cell. (b) Epithelial cell (E) associated with arteriole. L, lumen; arrow, endothelial cell; asterisk, smooth muscle cell; A, adventitial cell; Ly, lymphocyte. Panels (a) and (b) x2800.

 
Based on phenotypic similarities, it has been proposed that subcapsular and medullary TE are derived from a common ectodermal embryonic source that is distinct from the endodermal origin of cortical TE (21). Investment of pharyngeal pouch endoderm with a peripheral layer of pharyngeal cleft ectoderm during embryogenesis could easily account for the establishment of the subcapsular compartment epithelium (discussed in 22). The association of medullary TE with vascular elements suggests that neovascularization may be centrally involved in the initial organization of the medullary compartment. Invading vessels passing through the outer ectodermal layer into the endodermal mass could bring along some of the ectodermal epithelium or provide guidance for subsequent migration of ectodermal epithelial cells into the anlage of endodermal epithelium to form the medulla. Potential short-range interactions that could mediate initial organization of medullary TE include integrin-mediated recognition of extracellular matrix components associated with the vasculature (23) or the elaboration of soluble mediators such as epidermal growth factor (24) or members of the fibroblast growth factor family by endothelial, smooth muscle or fibroblastic vascular elements (25). Thymocyte-derived stimuli mediating the expansion and further differentiation of these `seeded' medullary TE cells would lead to the establishment of an organized medullary compartment. This organization of lymphoid compartments around vascular structures has a precedent in the spleen, where the white pulp is concentrically arranged around the central artery (26). While it is our working hypothesis that vascular elements serve as an organizing element for the medullary compartment, these data would also be consistent with a model whereby medullary stromal elements dictate the organization of the thymic vasculature.

The proximity of medullary TE with post-capillary venules, the most permeable segment of the vasculature, may be a significant feature of the thymic environment with important functional consequences. Analogous to the liver, where hepatocytes closest to portal venules are exposed to the highest concentrations of blood-borne nutrients from the gut (26), medullary TE adjacent to post-capillary venules would likely be exposed to high intrathymic levels of blood-borne self antigens or extra-thymic humoral mediators that could regulate medullary TE expression of self-antigens capable of mediating tolerance induction (27). In a reciprocal fashion, this location would also facilitate the access of chemokines produced by medullary TE to the local endothelium. This latter mechanism may contribute to the selective localization of peripheral T cells (19) or eosinophils (20) to the medullary compartment.


    Acknowledgments
 
This work was supported by grants (AI 24137 and AG04360) from the National Institutes of Health. We thank Dr Rudensky for critically reviewing the manuscript.


    Abbreviations
 
TE thymic epithelium

    Notes
 
Transmitting editor: M. J. Bevan

Received 10 March 2000, accepted 31 March 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Immunohistochemistry
 Results and discussion
 References
 

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