MICROSCOPY RESEARCH AND TECHNIQUE 38:227–236 (1997) Heterogeneity of Epithelial Cells in the Rat Thymus ERIC J. DE WAAL1 AND LOUK H.P.M. RADEMAKERS2* 1National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands of Pathology, University Hospital, Utrecht, The Netherlands 2Department KEY WORDS thymus; thymic epithelial cells; heterogeneity; immunohistochemistry; transmission electron microscopy ABSTRACT The morphological heterogeneity of the thymic epithelium has been well documented both at the light and electron microscopic level. Immunohistochemistry has revealed four broad classes of epithelial cells (EC): subcapsule/perivascular, cortical, medullary EC, and medullary Hassall’s corpuscles. Ultrastructural analysis has revealed further heterogeneity. In the cortex, four EC subtypes have been described ultrastructurally: subcapsular/perivascular, ‘‘pale,’’ ‘‘intermediate,’’ and ‘‘dark’’ EC. These subtypes are also present in the medulla. Two additional EC subtypes are restricted to the medulla: an undifferentiated subtype, and a subtype displaying signs of high metabolic activity. Based on the morphological features of the epithelium, it has been hypothetized that the thymic EC subtypes represent a process of differentiation. Microsc. Res. Tech. 38:227–236, 1997. r 1997 Wiley-Liss, Inc. INTRODUCTION The thymus is unique among the lymphoid organs in that the stromal compartment is composed largely of stationary epithelial cells (EC) (Schuurman, 1988). The thymic stroma contains among others two additional cell types: interdigitating dendritic cells (IDC) and macrophages (Mf) (Boyd et al., 1993; Duijvestijn and Hoefsmit, 1981; Kendall, 1981, 1991; Ushiki, 1986; Von Gaudecker, 1991). Immunohistochemically, thymic EC are characterized by the presence of cytoskeletal keratin intermediate filaments and, at the ultrastructural level, by the presence of tonofilaments and desmosomes (Bearman et al., 1978; Colic et al., 1989; Farr and Braddy, 1989; Hwang et al., 1974; Kampinga et al., 1989; Kendall, 1981, 1986, 1988; Laster et al., 1986; Singh, 1981; Van Haelst, 1967; Von Gaudecker, 1991; Von Gaudecker et al., 1986). The epithelium provides the three-dimensional framework, within which all other thymic cell types reside (Bearman et al., 1978; Hwang et al., 1974). Involvement in Thymocyte Differentiation Studies in the neonatal rat thymus have revealed that EC, IDC, Mf, and thymocytes preferentially occupy defined areas of the thymus, within which they are distributed and form cell complexes in a nonrandom manner (Brelinska et al., 1985). The cell composition of such complexes is typical and corresponds to the composition of multicellular complexes released after the dissociation of the thymic structure by enzymatic digestion as shown by experiments in mice (Brelinska et al., 1986; Toussaint-Demylle et al., 1991). Close interaction between the developing thymocytes and the thymic stroma is essential in intrathymic T cell differentiation (Boyd et al., 1993). This interaction is based on cell-cell plasma membrane contacts (Farr et al., 1985; Müller and Kyewski, 1993; Savion et al., 1989; Utsumi et al., 1991) and the secretion of soluble peptidic factors including cytokines by EC and other r 1997 WILEY-LISS, INC. thymic stromal cells (Denning et al., 1988; Geenen et al., 1992; Hadden, 1992; Kendall, 1991; Millington and Buckingham, 1992; Nabarra and Papiernik, 1991; Ritter and Boyd, 1993; Takahama et al., 1994; Wolf and Cohen, 1992). A key notion in thymology is that different stromal microenvironments govern distinct events in T-cell development (Boyd et al., 1993; Ritter and Boyd, 1993; Van Ewijk, 1988). Despite extensive research, however, the particular functions of individual stromal elements and their soluble mediators in T-cell development still are not well understood. To gain more insight into the heterogeneity of the thymic epithelium, we identified thymic EC subtypes ultrastructurally and studied their tissue distribution in the rat thymus. DISTRIBUTION OF EPITHELIAL CELLS IN THE THYMUS Cortical Epithelium EC in the subcapsular region form a layer of one or two cells deep. Slightly deeper in the cortex (i.e., in the outer cortex), thin, sheet-like EC are found displaying slender cytoplasmic processes. EC in the mid and deep cortex also have slender cytoplasmic processes providing a large surface area for cell-cell interactions between EC and developing thymocytes (Hoshino, 1963; Singh, 1981; Van Ewijk, 1988). Thymic Nurse Cells In the outer cortex, some EC appear to completely surround and enclose a large number (about 10–250) of thymocytes (Boyd et al., 1993). These so-called thymic nurse cells (TNC) have first been isolated from the mouse thymus by enzymatic digestion (Wekerle and Ketelsen, 1980; Wekerle et al., 1980). Later, TNC have *Correspondence to: L.H.P.M. Rademakers, Ph.D., Division of Histochemistry and Electron Microscopy, Department of Pathology, Academic Hospital Utrecht, P.O. Box 85,500, NL-3508 GA Utrecht, The Netherlands. Accepted in revised form 12 January 1995 228 E.J. DE WAAL AND L.H.P.M. RADEMAKERS been described in situ in the subcapsular and outer cortical regions of the human thymus (Dipasquale and Tridente, 1991; Ritter et al., 1981; Van De Wijngaert et al., 1983) and the mouse thymus (Andrews and Boyd, 1985; Kyewski and Kaplan, 1982; Leene et al., 1988; Van Ewijk, 1988). Thus far, TNC have not been recognized as such in the rat thymus (Ushiki, 1986). Hwang and coworkers (1974), however, have demonstrated epithelial-lymphoid cell complexes in the rat thymus at the ultrastructural level, which strongly resemble TNC. In the human thymus, TNC seem to be localized not only in the cortex, but also in the cortico-medullary junction and in the medulla (Dipasquale and Tridente, 1991). Similar observations have been reported in the mouse thymus by Kaneshima and coworkers (1987), although these authors point to the difficulty in identifying TNC in situ even by immunoelectron microscopy. Wekerle and coworkers (1980) have also stressed the difficulty to demonstrate TNC in situ, because, in the normal thymus, the epithelium is overcrowded with thymocytes. In addition, the visualization of TNC is hampered by the linkage of TNC to adjacent EC, resulting in the formation of an interlacing network of EC (Hiramine et al., 1990). TNC isolated from the mouse thymic medulla, in contrast to cortical TNC, contain only a small number of thymocytes (ToussaintDemylle et al., 1990). Scanning electron microscopic images of the mouse thymus have suggested that thymocytes migrate in or out these lympho-stromal complexes (Van Ewijk, 1988). TNC are thought to be a specialized microenvironment involved in T-cell differentiation (Aguilar et al., 1994; De Waal Malefijt et al., 1986; Dispasquale and Tridente, 1991; Kyewski, 1986; Lahoud et al., 1993). Although the epithelium is thought to be a sessile component of the thymus (Kendall, 1991), the subcapsular epithelium in particular may be a dynamic structure, since transitional forms have been observed between sheet-like EC, basket-like EC, and TNC depending on the type of cortical lympho-stromal interaction (Van Ewijk, 1988, 1991). TNC may therefore represent the end-stage of a continuum of such structures from open to completely closed (Boyd et al., 1993). Medullary Epithelium In contrast to the dendritic morphology of cortical EC, most medullary EC are closely packed displaying voluminous cytoplasm and short, blunt processes (Duijvestijn and Hoefsmit, 1981; Hoshino, 1963; Singh, 1981; Ushiki, 1986; Van Ewijk, 1988, 1991; Van Ewijk et al., 1988; Von Gaudecker and Müller-Hermelink, 1980). Whorls of highly keratinized medullary EC, so-called Hassall’s corpuscles (Kendall, 1981), are particularly well developed in the human thymus (Boyd et al., 1993; Lobach et al., 1985; Nicolas et al., 1989; Von Gaudecker and Schmale, 1974; Von Gaudecker et al., 1986). Depending on the strain involved, in rats and mice, these structures generally are rarely encountered and ill developed (Colic et al., 1988a; Duijvestijn and Hoefsmit, 1981; Hwang et al., 1974; Kendall, 1991; Lobach et al., 1987; Nicolas et al., 1989; Ushiki, 1986). Very little is known on the function of Hassall’s corpuscles. It has been postulated that they are graveyards for dead thymocytes (Boyd et al., 1993; De Maagd et al., 1985; Kendall, 1981; Von Gaudecker and Schmale, 1974; Von Gaudecker, 1986) or represent regions where unwanted antigens are accumulated (Kendall, 1981). ULTRASTRUCTURAL HETEROGENEITY Cortical Epithelium Recently, we have described four ultrastructurally identified EC subtypes in the cortex of the rat thymus and provided semiquantitative data on their tissue distribution (De Waal et al., 1993a,b). The morphological designation of EC subtypes was performed using the characteristics given in Table 1, the nuclear and cytoplasmic electron-density being the main parameters. The EC.1 subtype (Fig. 1a) is defined on its location in the subcapsular tissue. Within the EC.1 population, there is a heterogeneity of ‘‘pale,’’ ‘‘intermediate,’’ and ‘‘dark’’ cells, the intermediate manifestation being the most frequently occurring one. Such manifestations resemble either the pale EC.2 (Fig. 1b), intermediate EC.3 (Fig. 1c), or dark EC.4 (Fig. 1d). As the intermediate EC.3 cells (Fig. 1c) comprise a spectrum of cells ranging from relatively electron-lucent to electrondense, two additional parameters were used to distinguish these cells from pale EC.2 (Fig. 1b) and dark EC.4 (Fig. 1d). The round shape of the nucleus was a parameter to read pale cells on the one hand, and the irregularly shaped nucleus was used as a parameter to judge cells as intermediate and dark. Secondly, dark cells were separated from intermediate ones on the basis of the presence of an electron-dense ground substance. In the rat thymic cortex, the pale EC.2 was the most frequently occurring EC subtype comprising about 60% of the total EC number. Intermediate EC.3 were occasionally seen (about 25%), whereas dark EC.4 occurred in very low numbers only (about 5% of the total EC number). Due to the amorphous cytoplasmic ground substance, in these cells tonofilaments were sometimes hard to distinguish. Cytoplasmic vacuoles, however, could easily be recognized as a result of their clear appearance. As classical secretory granules are very rare in the thymic epithelium, such clear cytoplasmic vacuoles occurring in all EC subtypes are thought to be the terminal element of the secretory apparatus (Nabarra and Andrianarison, 1987b). These cytoplasmic vacuoles show a typical ultrastructural morphology: an electron-lucent matrix containing coarsely granular electron-dense material, often adhering to the inner face of the vacuolar membrane (Duijvestijn and Hoefsmit, 1981; Nabarra and Andrianarison, 1987a). All cortical EC subtypes were interconnected by desmosomes. Mostly mononucleate EC were seen, but binucleate pale EC.2 were observed in low frequency as well. Medullary Epithelium We have described the ultrastructural features of the medullary epithelium in the rat thymus as well (De Waal et al., 1996). The morphological designation of medullary EC subtypes (EC.1 to EC.6) was performed using the characteristics given in Table 2. EC.1 (Fig. 2a), EC.2 (Fig. 2b), EC.3 (Fig. 2c), and EC.4 (Fig. 2d) do not differ from analogous EC subtypes in the cortex. Their relative frequencies of occurrence are about 3% (EC.1), 36% (EC.2), 54% (EC.3), and 1% (EC.4), respectively. EC.5 (Fig. 2e) represents an undifferentiated EC subtype, which comprises only 1% of the total medullary EC population. In the medulla of the human 229 THYMIC EPITHELIAL CELL HETEROGENEITY TABLE 1. Main ultrastructural features of cortical epithelial cell (EC) subtypes in the rat thymus Subtype Designation Ultrastructural morphology Location EC.1 Subcapsular Continuous layer of cells bordering the thymic tissue beneath the capsule EC.2 Pale EC.3 Intermediate EC.4 Dark Low to high nuclear and cytoplasmic electron-density; heteroor euchromatic nucleus; prominent tonofilaments; rather well-developed Golgi complexes and RER; regularly electron-lucent cytoplasmic vacuoles Low nuclear and cytoplasmic electron-density; round, euchromatic nucleus; rather well-developed Golgi complexes and RER; inconspicuous tonofilaments; electron-lucent cytoplasmic vacuoles Moderate nuclear and cytoplasmic electron-density; euchromatic or heterochromatic nucleus of irregular shape; inconspicuous Golgi complex and tonofilaments; electron-lucent cytoplasmic vacuoles High nuclear and cytoplasmic electron-density; irregularly shaped, heterochromatic nucleus; electron-lucent cytoplasmic vacuoles; inconspicuous tonofilaments thymus, EC.5 are usually arranged in small groups (Van De Wijngaert et al., 1984), but in the rat thymic medulla they occur as solitary cells. The EC.6 subtype in the human thymus represents a large medullary EC with an electron-lucent cytoplasm, a round to oval euchromatic nucleus, prominent tonofilaments, welldeveloped rough endoplasmic reticulum, and small secretory vesicles (Van De Wijngaert et al., 1984). In the human thymic medulla, EC.4 and EC.6 are found in or around Hassall’s corpuscles (Van De Wijngaert et al., 1984). In the rat strain used in our studies, which lacks Hassall’s corpuscles in the thymic medulla, EC displaying a similar ultrastructure as the human EC.6 subtype were not present. However, large EC with low to moderate nuclear and cytoplasmic electron-density were seen (comprising 4% of the total population of medullary EC) either solitarily or in small groups, which showed a prominent cluster of large, electron-lucent cytoplasmic vacuoles, often carrying microvilli projecting into the vacuolar lumen. These vacuoles may contain flocculent material. In the rat classification, these cells were designated EC.6 (Fig. 2f). The first ultrastructural description of dark cells in the mouse thymus (and lymph nodes) was published by Izard and De Harven in 1968. These authors claimed that these so-called ‘‘dense reticular cells’’ were not part of the EC population. They however suggested a close relationship between ‘‘dark reticular cells’’ and EC, based upon the observation of many intermediate forms of variable size and density. More than a decade later, ultrastructural studies have confirmed the presence of pale and dark EC with reference to their nuclear and cytoplasmic electron density (Kendall, 1981; Singh, 1981). From an evolutionary point of view, differences in electron-density seem to be well conserved in the thymic epithelium as judged from observations in the thymus of the rainbow trout (Castillo et al., 1991) and the axolotl thymus (Tournefier et al., 1990). In the human thymus, six EC subpopulations have been described based on ultrastructural characteristics (Van De Wijngaert et al., 1984). This classification has been confirmed by other investigators for the human, mouse, and rat thymus (Boyd et al., 1993; Clarke and Kendall, 1989; Kendall, 1986, 1988, 1991; Kendall et al., 1988; Von Gaudecker, 1991). This (similar) heterogeneity has been claimed to hold for all vertebrate thymuses (Kendall, 1991). Our classification for the rat thymus indeed Scattered in the cortex, predominant in the outer cortex Scattered in the cortex, predominant in the mid and deep cortex Mid and deep cortex, predominant in the deep cortex closely resembles that of Van De Wijngaert and coworkers (1984) for the human situation. They designated four EC subtypes in the cortex and medulla of the human thymus. The first subtype is called EC.1, representing the subcapsular (cortex) and perivascular EC (medulla). The other three subtypes are defined by increasing grades of electron-density (i.e., pale EC.2, intermediate EC.3, and dark EC.4). As in the rat, a comparable heterogeneity (pale to dark forms) within the EC.1 population was present in the human thymus. Two further subtypes are exclusively found in the medulla: EC.5 showing the ultrastructural characteristics of undifferentiated elements, and EC.6 displaying signs of high metabolic activity (Van De Wijngaert et al., 1984). The cortical pale EC.2, intermediate EC.3, and dark EC.4 show a gradation in representation from the outer subcapsule to the inner cortex (Boyd et al., 1993; Van De Wijngaert et al., 1984). In the human thymus, pale EC.2 and intermediate EC.3 in the cortex probably contribute to TNC (Boyd et al., 1993; Kendall, 1991; Van De Wijngaert et al., 1984; Von Gaudecker, 1991). In contrast to the human thymus (Van De Wijngaert et al., 1984), the ultrastructural features of EC.2, EC.3, and EC.4 in the cortex of the rat thymus are slightly different from those characterizing their medullary counterparts (Tables 1, 2). In particular, tonofilaments are more prominent in medullary EC.2 and EC.3 than they are in cortical EC.2 and EC.3. Also, EC.6 in the medulla of the rat thymus has a completely different ultrastructure in comparison with the human situation. These cells have been designated as ‘‘cystic’’ (Mandel, 1970), ‘‘hypertrophic’’ (Bennett, 1978), or ‘‘alveolar layrinthine’’ EC (Nabarra and Andrianarison, 1987b). Probably, this EC subtype is involved in thymic hormone production (Bennett, 1978; Mandel, 1970). IMMUNOHISTOCHEMICAL HETEROGENEITY To elucidate the structural organization of the thymic epithelium, many immunohistochemical and immunoelectron microscopic studies have been conducted (for references see Boyd et al., 1993). Extensive panels of monoclonal antibodies have revealed complex distribution patterns of keratin subtypes typical of simple and stratified epithelia (Colic et al., 1988a, 1989; Farr and Braddy, 1989; Gay-Bellile et al., 1986; Laster et al., 1986; Nicolas et al., 1985, 1989; Savino and Dardenne, 1988; Von Gaudecker et al., 1986). Immunostaining 230 E.J. DE WAAL AND L.H.P.M. RADEMAKERS Fig. 1. Electron micrographs of the various cortical epithelial cell subtypes. a: EC1. Subcapsular epithelial cell (EC) showing prominent tonofilaments (arrows). Asterisk: Capsular connective tissue; arrowhead: Golgi complex; double arrow: basement membrane. 38,200. Inset: Detail of a cluster of electron-lucent vacuoles containing vesicular structures which are regularly observed in subcapsular EC. 314,000. b: EC2. Pale EC having an extensive cytoplasm containing numerous polyribosomes. Desmosomes (arrows) are regularly present. Arrowhead: Golgi complex. 35,200. c: EC3. Intermediate EC (asterisk) having a heterochromatic nucleus and a low amount of cytoplasm. Arrows: lysosomes; arrowhead: cytoplasmic vacuole. 39,500. d: EC4. Dark EC with cytoplasmic vacuoles (arrow) and dilated cisterns of the rough endoplasmic reticulum (arrowheads). Large arrow: bundle of tonofilaments. 37,200. THYMIC EPITHELIAL CELL HETEROGENEITY TABLE 2. Main ultrastructural features of medullary epithelial cell (EC) subtypes in the rat thymus Subtype Designation EC.1 Perivascular EC.2 Pale EC.3 Intermediate EC.4 Dark EC.5 Undifferentiated EC.6 Cystic Ultrastructural characteristics Located around capillaries; low to high nuclear and cytoplasmic electrondensity; nucleus round or irregularly shaped, hetero- or euchromatic; prominent tonofilaments; electronlucent cytoplasmic vacuoles Low nuclear and cytoplasmic electrondensity; round, euchromatic nucleus; poorly to moderately developed tonofilaments; electron-lucent cytoplasmic vacuoles Moderate nuclear and cytoplasmic electron-density; irregularly shaped nucleus, hetero- or euchromatic; inconcpicuous, moderately developed tonofilaments; electron-lucent cytoplasmic vacuoles High nuclear and cytoplasmic electrondensity; nucleus of irregular shape, hetero-chromatic; inconspicuous tonofilaments; few cytoplasmic vacuoles Low nuclear and cytoplasmic electrondensity; round, euchromatic nucleus; inconspicuous tonofilaments; polyribosomes Low to moderate nuclear and cytoplasmic electron-density; eccentric, round or irregularly shaped heteroor euchromatic nucleus; rather welldeveloped rough endoplasmatic reticulum; small tonofilaments; large intra-cytoplasmic cysts with intraluminal microvilli with anti-keratin antibodies has revealed a different topography of labeled EC in rat, mouse, and human thymus (Meireles De Souza et al., 1993). This group observed an important similarity in the topography of specific keratin subclasses in species of the same mammalian order. Other antigenic determinants with an unknown functional significance demonstrated complex staining patterns as well (Boyd et al., 1993; Colic et al., 1988b; De Maagd et al., 1985; Izon and Boyd, 1990; Kampinga et al., 1987, 1989; Kaneshima et al., 1987; McFarland et al., 1984; Rouse et al., 1988; Von Gaudecker et al., 1986, 1989). Yet, four broad classes of epithelium have been identified phenotypically: subcapsule/perivascular, cortical, medullary EC, and medullary EC forming Hassall’s corpuscles (Boyd et al., 1993; Janossy et al., 1986; Kampinga et al., 1989; McFarland et al., 1984; Van Ewijk, 1991). Many monoclonal antibodies raised against thymic EC react either with subcapsular and medullary epithelium on the one hand or with cortical epithelium on the other hand (Boyd et al., 1993; Colic et al., 1988b; Farr et al., 1991; Haynes et al., 1983, 1984; Izon and Boyd, 1990; Kampinga et al., 1987; Kaneshima et al., 1987; Lampert and Ritter, 1988; Pavlovic et al., 1993; Von Gaudecker, 1991). Some antibodies to EC detect epitopes that are also present on thymocytes (Godfrey et al., 1990) or IDC (Izon et al., 1994). Based on distribution patterns of keratin expression in the hamster thymus, it has been suggested that phenotypic markers of EC heterogeneity should be considered as indicators of a continuous plasticity of the 231 epithelium, rather than determinants to define definitive EC subsets (Lannes Vieira et al., 1994; Meireles De Souza and Savino, 1993). The subcapsular and medullary epithelium constitute the endocrine component of the thymus (Auger et al., 1987; Geenen et al., 1992; Von Gaudecker et al., 1986; Von Gaudecker, 1991), although, in the mouse and human thymus, thymic hormones have been localized in cortical EC as well (Auger et al., 1984; Jambon et al., 1981; Savino and Dardenne, 1984; Schuurman et al., 1985). Neuroendocrine markers are also preferentially distributed in the subcapsular and medullary epithelium of the human thymus. Scattered in the cortex, solitary EC express such markers as well (Geenen et al., 1987; Moll et al., 1988). Whereas all EC express MHC class I products, MHC class II antigens are differentially expressed in the cortical and medullary epithelium. Cortical EC are strongly MHC class II-positive in mice (Farr and Nakane, 1983; Farr and Sidman, 1984; Nabarra and Papiernik, 1988; Rouse et al., 1979; Van Ewijk et al., 1980), rats (Kendall et al., 1990) and humans (Hamblin and Edgeworth, 1988; Ritter et al., 1981; Van Baarlen et al., 1989; Von Gaudecker et al., 1986). The extent of MHC class II immunolabeling in the medullary epithelium varies between different species (Ritter and Crispe, 1992). In mice, a large proportion of the medullary EC expresses MHC class II molecules (Farr et al., 1993; Farr and Nakane, 1983; Nabarra and Papiernik, 1988; Rouse et al., 1979; Surh et al., 1992; Van Ewijk, 1991; Van Ewijk et al., 1980; Van Ewijk, 1984). In contrast, MHC class II immunolabeling is far less prominent or even absent in the medullary epithelium of the rat (Kendall et al., 1990; Schuurman et al., 1990) and human thymus (Hamblin and Edgeworth, 1988; Haynes, 1984; Van Baarlen et al., 1989; Von Gaudecker et al., 1986; Von Gaudecker et al., 1997). The subcapsular epithelium may express MHC class II antigens in mice (Farr et al., 1992) and humans (Haynes, 1984; Janossy et al., 1986; McFarland et al., 1984). CONCLUDING REMARKS Early light microscopic studies in mice (Cordier and Haumont, 1980) have suggested that cortical EC are ectodermal and subcapsular/medullary EC endodermal in origin. Early studies suggested that subcapsular/ perivascular and pale EC in the cortex and medulla of the human thymus are descendants of the ectoderm, whereas intermediate and dark EC in the medulla and inner cortex come from the endoderm (Norris, 1938; Von Gaudecker, 1986). Recent immunohistological studies, however, have questioned the dual origin of the thymic epithelium, suggesting that there is a single epithelial stem cell that can give rise to the many different EC subpopulations during thymic development (Boyd et al., 1993; Lampert and Ritter, 1988; Kendall, 1991; Ritter and Boyd, 1993; Von Gaudecker, 1991). This hypothesis is supported by the presence of EC having antigenic characteristics of both cortical and subcapsular/medullary epithelium in the involuted thymus of young children after a period of acute illness (Van Baarlen et al., 1989), some thymomas (Lampert and Ritter, 1988; Willcox et al., 1987) and cultured thymic fragments implanted in athymic (nude) rats (Kendall et al., 1988). Early ultrastructural observa- 232 E.J. DE WAAL AND L.H.P.M. RADEMAKERS Fig. 2. Electron micrographs of medullary epithelial cell subtypes. a: EC1. Perivascular EC with elongated cytoplasmic extensions bordering connective tissue (asterisk) surrounding a capillary. Desmosomes (arrows) are well developed. Arrowhead: basement lamina of EC. 39,200. b: EC2. Pale EC with voluminous cytoplasm having a high density of polyribosomes. Golgi complex and RER are well developed. Vacuoles with amorphous content (arrows). 37,000. c: EC3. Intermediate EC with moderate cytoplasmic and nuclear electron density. Tonofilaments in connection with well-developed desmosomes are present (arrows). 35,800. d: EC4. Dark EC having an extremely dilated RER and cytoplasmic vacuoles. 36,200. e: EC5. Undifferentiated EC containing a high density of polyribosomes and distinct tonofilaments (arrows). 39,000. f: EC6. Cystic EC having well-developed rough endoplasmic reticulum. Note a cluster of intracytoplasmic cysts (arrowhead). Some intraluminal microvilli are present (arrow). 35,400. THYMIC EPITHELIAL CELL HETEROGENEITY Fig. 2. tions in the rat thymus have suggested the possible existence of one lineage of EC as well (Hwang et al., 1974). This issue will be further discussed by Von Gaudecker and coworkers, and by Röpke et al. (this issue). Immunohistochemical studies have provided a basis to postulate that thymic EC differentiate through phenotypically discrete stages in vivo (Nicolas et al., 1989). Early ultrastructural observations on the mouse thymus have suggested a sequential differentiation of EC as well (Mandel, 1970). In this hypothesis, medullary EC ultimately form Hassall’s bodies by terminal differentiation (Laster et al., 1986; Lobach et al., 1985, 1987; Nicolas et al., 1989). Human Hassall’s corpuscles reveal a striking similarity to keratinizing squamous epithelium in the epidermis (Laster et al., 1986; Von Gaudecker and Schmale, 1974). Similarities between the thymic medulla and the epidermis have been postulated based on antigenic cross-reactivities as well (Schmitt et al., 1987). From patterns of keratin distribution within the human thymus, Laster and coworkers (1986) suggested that thymic EC undergo a pathway of differentiation similar to that observed in epidermal keratinocytes. Based on the ultrastructure of the human thymic epithelium, Van De Wijngaert and coworkers (1984) likewise have postulated that morphologically distinct EC subtypes represent sequential stages in a differentiation process. Our recent ultrastructural studies on alterations of the rat thymic epithelium after chemical exposure suggest a similar phenomenon (De Waal et al., 1993b). 233 (Continued.) ACKNOWLEDGMENTS Henk-Jan Schuurman (Sandoz Pharma Ltd, Basel, Switzerland), Henk van Loveren and Joseph G. 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