SEARCH

SEARCH BY CITATION

Keywords:

  • endocrine-like cells;
  • piecemeal degranulation;
  • thymus;
  • chick embryo;
  • electron microscopy

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

The endocrine-like cells of the chick embryo thymus were studied by transmission electron microscopy and a highly characteristic pattern of cell secretion referred to as piecemeal degranulation (PMD) was identified. This is the first description of PMD in embryonic cells. © 2005 Wiley-Liss, Inc.

The term “piecemeal degranulation” (PMD) has been coined in the early 1970s by Dvorak et al. to describe a unique secretory process recognized in basophils, mast cells, and eosinophils, and ultrastructurally characterized by a slow and focal discharge of granule-stored material without granule opening to the cell exterior (Dvorak, 1991).

For about 30 years, the concept of PMD has been solely restricted to these three types of secretory cells. Recently, we have identified ultrastructural features highly suggestive of PMD in other kinds of cells, namely, the endocrine cells of the human and murine gastrointestinal tract and the chromaffin cells of the mouse and rat adrenal medulla (Crivellato et al., 2002, 2003a, 2004a). These results prompted us to speculate that PMD might represent a general secretory pattern, alternative to exocytosis, in granulated cells involved in paracrine/endocrine functions (Crivellato et al., 2003b).

Identification of PMD lies on precise ultrastructural criteria (Dvorak, 1991). Cells undergoing PMD indeed present characteristic admixture of resting granules, granules with various extent of content losses, and empty granules. Notably, all granules maintain their individual structure and do not fuse with each other or with the plasma membrane. Release of stored material by PMD may be visualized as pieces or packets of lost granule particles, which leave focal areas of electron luciencies inside involved granules.

As secretory granules do not open to the cell exterior during the PMD reaction, it has been proposed that the discharge mechanism implies the formation of vesicles that shuttle back and forth between the granules and the plasma membrane (Dvorak, 1994). Vesicles containing bits of granule contents would bud from the perigranule membrane, move through the cytoplasm, and fuse with the plasma membrane. Parallel formation of endocytotic vesicles from the plasma membrane would fuse with the granule membrane in order to maintain the granule size. If the rate and amount of vesicular traffic are balanced, granule containers empty in a piecemeal fashion but maintain a constant size. If, on the other hand, the inward flow of endocytotic vesicles exceeds the outward flow of exocytotic vesicles, granule chambers become enlarged (Dvorak, 1994).

In the course of a recent ultrastructural investigation on mast cell development in the chick embryo thymus (data not shown), we have been struck by the impressive PMD morphologies expressed by endocrine-like cells (ECs) in the gland rudiments. Therefore, we decided to undergo a systematic search for PMD ultrastructural details in these cells.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Fertilized White Leghorn chick eggs were incubated under conditions of constant humidity at 37°C and utilized from day 13 to hatching. Thymic tissues from the third to the fifth lobes were bilaterally excised and fixed by immersion in 2% paraformaldehyde and 3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 3 hr, postfixed in phosphate-buffered 1% osmium tetroxide for 1.5 hr, dehydrated in graded ethanol series, and embedded in Epon 812. Sections were counterstained with uranyl acetate and lead citrate and examined in a Philips CM 12 electron microscope at 80 kV.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

A heterogeneous population of ECs could be found in the chick embryo thymus from day 15 onward. These cells were located in both the outer (Figs. 1 and 2) and deep (Figs. 3 and 4) medulla. All ECs contained numerous cytoplasmic membrane-bound secretory granules, which presented different size, shape, and electron density. This allowed ECs to be easily distinguished from thymic epithelial cells. Some granules were round or elliptic, 270–360 nm diameter, and highly electron-dense (Figs. 3 and 4). Other were either round-oval, 0.6–1.3 μm in diameter, densely packed, and highly electron-dense (Fig. 1) or round, moderately electron-dense, with a diameter of 0.6–1.2 μm (Fig. 2).

thumbnail image

Figure 1. Electron micrographs illustrating PMD features in EC of the chick embryo thymus at day 15. This EC shows irregularly shaped electron-dense granules intermingled with enlarged empty containers (small arrows) and granules containing dissolved matrices (large arrows). Scale bar = 1 μm.

Download figure to PowerPoint

thumbnail image

Figure 2. Electron micrographs illustrating PMD features in EC of the chick embryo thymus at day 19. This EC presents an admixture of round, moderately electron-dense granules (large arrows), haloed granules (arrowheads), and empty containers (empty arrows). One of them exhibits a bud-like projection (thin arrow). Scale bar = 1 μm.

Download figure to PowerPoint

thumbnail image

Figure 3. Electron micrographs illustrating PMD features in EC of the chick embryo thymus at day 15. These ECs show round or elliptic electron-dense resting granules (large arrows) intermingled with enlarged piecemealed or haloed granules that show mobilized content materials (thin arrow). Electron-dense and -lucent vesicles are seen (small arrows). At the end of the secretory process, granules present as dilated empty containers (double arrow). Scale bar = 300 nm.

Download figure to PowerPoint

thumbnail image

Figure 4. Electron micrographs illustrating PMD features in EC of the chick embryo thymus at day 16. These ECs show round or elliptic electron-dense resting granules (large arrows) intermingled with enlarged piecemealed or haloed granules that show mobilized content materials (thin arrows). Numerous electron-lucent vesicles and vesicles loaded with dense material are seen either free (small arrows) or clustered (empty arrow) in the cytoplasm. At times, vesicles bud from the surface of the granules in a chain-like fashion (double arrows). Granules may exhibit strictures or tail-like features (curved arrow). Scale bar = 300 nm.

Download figure to PowerPoint

Viewed at low/middle magnification, the majority of ECs exhibited an admixture of resting unaltered granules and dilated, nonfused, variably emptied granules (Figs. 1–4). Resting granules could be identified by their compact structure and closely adhering limiting membrane. Enlarged granules showed variable mobilization of their secretory content, leaving haloed (Figs. 2 and 4) or piecemealed (Fig. 3) patterns. In some instances, granules exhibited a diffuse decrease of electron density due to uniform loss of content material (Fig. 2). Remarkably, a proportion of granules transformed into large empty containers (Figs. 1–3).

At higher magnification, altered granules presented bud-like or tail-like protrusions of their limiting membrane (Figs. 2 and 4). An impressive number of membrane-bound vesicles with a diameter of 50–150 nm was observable in the cytoplasm of some ECs (Figs. 3 and 4). Most vesicles were electron-lucent but many exhibited an inner electron-dense content similar in structure and density to the secretory material stored in the granules. Vesicles were free in the cytoplasm but some of them were grouped in clusters (Fig. 4). Very often, they appeared as fused with the perigranule membrane, in a process of attaching to or budding from the granule (Figs. 3 and 4); at times they formed unique chain-like structures, which remained adherent to the granule-limiting membrane (Fig. 4). All ultrastructural features here described were recognized during the whole examined embryonic period but with particular frequency from day 14 to 16; they showed a tendency to decrease during the following days. ECs exhibiting PMD were found both in the outer and inner medulla; their density apparently diminished near hatching. Exocytosis was rarely observed in EC.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

In the present study, we document for the first time the existence of PMD morphologies in an embryonic organ. ECs in the chicken thymus are highly characteristic granulated cells that store 5-hydroxytryptamine and possibly peptides in their secretory granules (Ciaccio, 1942; Reggiani, 1946; Frazier, 1973; Häkanson et al., 1974). ECs are ultrastructurally distinct from thymic epithelial cells since they represent two different cell types (Kendall, 1991). According to literature, ECs are widely distributed in the medulla, increase in number at week 2 and between weeks 9 and 20 after hatching, but their origin and biological significance are poorly understood (Häkanson et al., 1974; Chan, 1994). They show images of exocytosis (Chan, 1994), which indicate that these cells are actively secreting, although the nature of the product(s) is not clear.

The data here reported favor the view that these cells may release their granule content not only by exocytosis but also by PMD. Ultrastructural data indeed fulfill the diagnostic criteria for PMD (Dvorak, 1991). In fact, ECs exhibit high granule polymorphism (admixture of resting granules, dilated altered granules, and empty containers); absence of granule fusion; characteristic haloed patterns of residual secretory content; great amount of electron-dense and -lucent vesicles, free in the cytoplasm or attached to granules; and multiple features of vesicles budding from the granule-limiting membranes.

A role for PMD pathways in EC of embryonic thymus could be suggested according to the recently proposed mechanisms of central tolerance induction (Crivellato et al., 2004b). It is generally accepted that the thymic medulla is the main site for T-cell-negative selection (Sprent and Kishimoto, 2002). It has been recognized that this thymic compartment contains stable groups of cells displaying the anatomical organization, morphology, and functional activity of other tissues, namely, Hassal's bodies (squamous epithelial cells that resemble epidermal epithelium), cystic organoid structures highly resembling respiratory epithelium, neuroendocrine cells, myoid cells, and solitary thyroid follicles (Farr et al., 2002). According to most recent views, this characteristic microanatomy and phenotypic expression maintain the spectrum of peripheral self in the thymus (immunological homunculus) with profound implications in the mechanisms of T-cell central tolerance and susceptibility to organ-specific autoimmunity (Kyewski et al., 2002). ECs may be part of the complex mosaic of structural and antigenic heterogeneity in the thymus. They represent indeed a composite cell population possibly releasing different secretory products. Through quantal liberation of granule-stored material by PMD, they are well endowed to provide the right antigen(s) concentration to induce tolerance (Durkin and Waksman, 2001).

In conclusion, this study provides for the first time ultrastructural description of PMD in embryonic secretory cells. This finding underlines the concept that PMD has a broader spectrum of expression than hitherto reported. It may represent indeed a general model of granule release alternative to exocytosis in secretory cells involved in endocrine-paracrine functions.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED