By
§
From the * Department of Biological Structure, the Department of Immunology, and the § Howard
Hughes Medical Institute, University of Washington, Seattle, Washington 98195-7420
The thymus is organized into discrete subcapsular, cortical, and medullary environments that have been defined by the distribution of morphologically and phenotypically distinct populations of epithelial cells and other
stromal cell constituents (1). T cell progenitor cells entering the thymus move in a centripetal fashion from the
subcapsular region through the cortex to the medullary
area during their maturation process. Proximal stages of
thymocyte development thought to occur in the subcapsular and cortical compartments include expansion of the
progenitor pool and expression of pre-TCR, which in turn
selects thymocytes with a functional TCR- Concurrent with this process of positive selection, self-reactive thymocytes are eliminated. The anatomic setting
for this process within the thymus is variable and appears to
be dependent on the qualitative and quantitative parameters of ligand expression. Extensive cortical deletion has
been observed in some TCR transgenic mouse models,
whereas in others deletion was more evident in corticomedullary and medullary compartments (7). Hematogenously
derived dendritic cells localized to the inner cortex and
corticomedullary interface represent one major cell type able
to mediate clonal deletion in the thymus. Several transgenic
model systems have also pointed to a role for medullary
thymic epithelium in this process (8), although the extent of functional overlap of these different cell types in
their ability to mediate negative selection remains unclear.
A significant constraint on the process of thymic negative selection for a given self-protein is a requirement for
expression of the cognate peptide-MHC complex by thymic APCs, either as a consequence of expression of this
particular protein by the thymic APCs or due to adequate
levels in the circulation. Although such a requirement
would be compatible with negative selection of T cells specific for some self molecules, it poses a challenge for thymic
elimination of T cells specific for self-proteins expressed in
developmentally, temporally, or spatially regulated patterns, such as inducible tissue-specific proteins, hormones that
exhibit oscillating levels in the circulation, or proteins
whose pattern of expression are under tight developmental
control. It has been proposed that T cells reactive to such
autoantigens are inactivated in the periphery (11).
An alternative mechanism of central tolerance to one such
autoantigen is reported in this issue (12). Klein et al. examined the mechanism of CD4 T cell tolerance to C-reactive
protein, a prototype inducible serum protein produced in
the liver during acute phase reaction. In this study, mice expressing human C-reactive protein (hCRP) transgene under autologous regulatory elements were crossed with two
strains of mice expressing transgenic TCRs specific for
dominant and subdominant hCRP epitopes. Based on their
initial observation of a profound deletion of CD4+ thymocytes in these mice irrespective of hCRP serum levels,
the authors examined tissue specificity of transgene expression and found hCRP mRNA in medullary thymic epithelial cells in addition to hepatocytes. The physiological relevance of hCPR expression by medullary thymic epithelium
(TE) was supported by similarities in the regulation of
hCRP expression by medullary TE and hepatocytes and by
the demonstration of CRP message in human medullary
thymic epithelium and homologous acute phase proteins in
medullary thymic epithelium in mice. The demonstration
by Klein et al. that the murine homologue of human CRP
is expressed by medullary TE in a physiologically regulated
manner, together with previous studies indicating thymic
expression of a wide spectrum of molecules expressed by
other organs and tissues of ectodermal or endodermal origin, indicate that expression of extrathymic "tissue-specific"
genes in the thymus is more prevalent than previously appreciated and is of importance for the central tolerance induction.
The association of numerous neuroendocrine peptides
such as vasopressin and oxytocin with thymic epithelial
cells in situ (13), and the production of some of these hormones by thymic epithelial cell lines in vitro (14), has led
to the consideration of the thymus as an endocrine organ.
Additional proteins that are normally associated with other
tissues/cell types and have been localized to the thymus at
the mRNA level include insulin (15), interphotoreceptor
binding protein (16), and myelin basic protein (17), as well
as the precursor of peptide amidating enzymes necessary to
generate biologically active regulatory peptide hormones
(peptidyl-glycine a-amidating monooxygenase [PAM]; reference 14). Because of the diversity of these "tissue-specific" molecules detected within the thymus and the observation that thymic and hepatic CRP expression appears to
be similarly regulated (12), thymic expression of these
genes may reflect a general physiological property of medullary thymic epithelium. Thus, as a consequence, medullary thymic epithelium displays within the thymus a mosaic of epithelial molecules also expressed by other epithelial tissues and organs.
Epithelial cells comprising the medullary compartment
exhibit considerably more morphological heterogeneity
than do their cortical counterparts. Several distinctive epithelial cell types found in the medulla are depicted in Fig.
1. One subset is associated with basal laminae of major
medullary blood vessels, but continue out through the cortex along connective tissue septae and are continuous with
subcapsular epithelial cells that share the basal lamina of the
thymic capsule. These cells can be identified by their reactivity with the 8.1.1 mAb, which detects a 36-KD cell surface molecule (18). Additional medullary epithelial subsets ultrastructurally resemble other types of terminally differentiated epithelium, including whorls of stratifying epithelium reminiscent of ectodermally derived epidermal epithelium (Hassal's bodies), ciliated epithelium, similar to that
observed in proximal segments of the respiratory system
and in the reproductive system derived from endoderm
(19), and epithelial cells containing small electron-dense vesicles resembling neurosecretory cells found in endocrine
organs and the diffuse neuroendocrine system (4). Phenotypic heterogeneity of medullary TE has also been well
documented, with shared antigenicity with a variety of cell
types, including keratinocytes (20), neuroendocrine cells
(21), and neural crest cells (22). Medullary epithelium has
recently been shown to express class Ib HLA-G molecules, which are also expressed by fetal trophoblasts (23).
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chain to undergo further maturation and to become CD4+8+ (double
positive, DP) thymocytes expressing low levels of TCR-
/
. Further development of DP thymocytes is dependent on
recognition of MHC-peptide ligands expressed by cortical
thymic epithelial cells. DP thymocytes expressing receptors
of appropriate specificity for these ligands exhibit some
phenotypic characteristics of activation and downregulate
expression of either CD4 or CD8 to yield mature CD4+ or
CD8+ single positive thymocytes (6).
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Fig. 1.
Distinctive epithelial components of the medullary
thymic environment. (a) Epithelial cells lacking ultrastructural
features of terminally differentiated epithelium and associated
with basal laminae associated
with major blood vessels in the
medulla. These cells morphologically and phenotypically resemble subcapsular epithelium. (b)
Hassal's bodies consisting of concentric whorls of stratified keratinizing epithelium. Well developed in human thymus, they are
small and relatively sparse in the
murine thymus. (c) Ciliated columnar epithelium is often associated with
cystic structures in the medulla. Occasional cells possess large ciliated vacuoles. (d) Epithelial cells considered to be neuroendocrine, based on ultrastructural similarities of small electron-dense cytoplasmic granules with
those observed in bona fide neurosecretory cells. These cells often have
distinctive cytoplasmic vacuoles with microvilli projecting into the lumen
of the vacuoles.
What is the mechanism underlying the expression of "extrathymic" gene products by thymic epithelial cells and how is this expression regulated? Although a generalized derepression or "loose" transcriptional control of genes normally expressed by differentiated epithelial cells of different lineages cannot be excluded, the regulated nature of CRP expression by medullary TE as reported by Klein et al. (12) would argue against such a mechanism. We favor the hypothesis that expression of these molecules reflects differentiation of medullary TE along different epithelial lineages. If expression of differentiated epithelial gene products reflects differentiation of medullary TE along different lineage-restricted pathways, these proteins should be heterogeneously expressed with different gene products associated with distinct subsets of medullary thymic epithelium. Consistent with this hypothesis, thymic expression of PAM is restricted to small scattered foci of medullary TE (14), as are cells expressing high molecular weight keratins associated with terminally differentiated keratinocytes (24).
Expression of these molecules may represent the intrinsic
developmental potential of medullary thymic epithelium,
perhaps reflecting their embryological derivation. A widespread distribution of endocrine cells throughout other tissues and organs has become increasingly apparent with the
development of appropriate antibody and nucleic acid reagents, although the embryological origins of these cells,
their developmental history, and function remain poorly
understood. Another nonexclusive possibility is that local stimuli generated within the thymus play an important role
in this process. Previous studies have demonstrated that interactions between thymocytes and thymic stromal cells
control not only T cell development and selection but also
medullary epithelial cell development and the organization
of the medullary compartment. The medullary compartment is present but fails to organize properly in mutant
mice unable to productively generate CD3+ thymocytes
(25) and is also disrupted in some lines of TCR transgenic
mice (26). One aspect of medullary epithelial development affected by these interactions may be the generation of
medullary TE expressing phenotypic, morphologic, and/or
functional properties usually associated with other epithelial
tissues or organs. In this case, the generation of CD3+ T
cells required for the organization and differentiation of medullary TE and the resulting expression of "extrathymic" epithelial molecules by these cells could provide positive feedback to facilitate the clonal elimination of thymocytes with reactivity to the epithelial antigens expressed
with the thymus. Circumstantial evidence that supports this
possibility is the observation that a small population of
medullary epithelial cell identified with the 10.1.1 mAb in normal murine thymi (27) is lacking in the poorly organized medullary thymic compartment of SCID or RAG/
mice (Farr, A., unpublished observations). The high levels
of PAM expressed by 10.1.1+ thymic epithelial cells and
the ability of these cells to secrete an amidated peptide hormone, oxytocin, which is classically associated with the hypothalamohypophyseal tract (14), would indicate that these
cells represent a subset of epithelial cells that have differentiated along a neuroendocrine pathway. A more extensive
analysis of the cellular composition of the medullary TE in
these mutant mice should indicate the extent to which normal T cell development affects medullary expression of
"extrathymic" epithelial gene products.
Expression of the "extrathymic" gene products by thymic medullary epithelium alone does not warrant presentation of peptides derived from these proteins by MHC class
II molecules. As suggested by Klein et al., secreted proteins
such as CRP can be taken up and presented by professional
APCs, namely thymic dendritic cells (12). However, cellular proteins which are not secreted or are secreted at a very
low level by thymic medullary cells are unlikely to be presented by thymic DC. Can medullary epithelial cells themselves induce tolerance in developing thymocytes? Experiments using chimeric transgenic mice with a restricted
expression of MHC ligands in thymic medulla revealed tolerance induction by both deletional and nondeletional
mechanisms with a variety of different antigens (8).
Furthermore, medullary epithelial cell lines induced with -IFN are able to process and present exogenous protein
antigens in an MHC class II-restricted fashion (28, 29). A
closer examination of the antigen-presenting properties of
medullary epithelial elements in situ and analyses of ex vivo
isolated cells revealed significant degree of heterogeneity.
Only a subset of medullary epithelial cells express MHC
class II molecules (1, 30). These cells are apparently capable
of processing and presenting at least some endogenous proteins as suggested by staining with mAbs specific for a major self peptide-MHC class II complex (31). However, class II-positive medullary epithelial cells differ significantly in their antigen-presenting properties as evidenced by highly variable expression of MHC class II bound invariant chain
peptide CLIP, an essential intermediate of MHC class II
processing (31). Finally, only a subset of medullary epithelial cells expresses the CD28 ligand B7 (9, 32), although the
functional significance of CD28-B7 interaction in negative
selection of thymocytes remains unclear.
Thus, thymic medullary epithelium is composed of diverse epithelial elements with different antigen-presenting potential and that display a mosaic of "peripheral" self-proteins. The functional importance of thymic expression of such proteins in tolerance induction is indicated by recent observations linking the level of expression of such autoantigens in the thymus and the predisposition to related autoimmune diseases (16, 32, 33). Further experimentation is needed to understand the role of specific subsets of thymic medullary epithelial cells in establishment of the central tolerance to peripheral autoantigens with restricted tissue distribution or inducible expression.
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Footnotes |
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Address correspondence to Andrew G. Farr, University of Washington, School of Medicine, Department of Biological Structure, Box 357420, Seattle, WA 98195-7420. Phone: 206-685-1584; Fax: 206-543-1524; E-mail: farr{at}u.washington.edu
Received for publication 13 May 1998 and in revised form 18 May 1998.
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