The examined tonsils play an important role in the defensive mechanisms against foreign pathogens (Surjan,1987; Scadding,1990; Perry and Whyte,1998). Early in 1884, Von Waldeyer-Hartz first reported the presence of lymphoid tissue in the human pharynx, and described its specific arrangement as a ring of lymphoid tissue, now termed Waldeyer's ring. The ring is composed of the nasopharyngeal tonsil (NT), or adenoid, attached to the roof of the nasopharynx, the paired tubal tonsils (TT) situated at the nasopharyngeal openings of the eustachian tubes, the paired palatine tonsils (PT) positioned in the oropharynx and the lingual tonsil (LT) on the posterior third of the tongue (Perry and Whyte,1998).
In recent years, many studies have examined the histological or ultrastructural features of tonsils in various animal species (Kumar and Timoney,2005a,2005b,2005c,2005d; Casteleyn et al.,2007; Breugelmans et al.,2011), such as horse, sheep, rabbits, and dog (Belz and Heath,1995a; Gebert,1995; Gebert et al.,1995; Casteleyn et al.,2008; Brandtzaeg,2011). Macroscopic examinations from these studies revealed that tonsils were always covered by specialized epithelia. However, the histology and ultrastructure of porcine tonsils are still poorly documented. Therefore, in this study, we investigate the morphology and ultrastructure of four porcine tonsils (the tonsil of the soft palate, the paraepiglottic tonsil, the pharyngeal tonsil, and the TT) to gain a better understanding of their immune functions.
MATERIALS AND METHODS
Twenty pigs of either sex aged 5 months were raised in the farm of the Nanjing Agricultural University and were euthanized with a lethal intravenous injection of pentobarbital sodium (100 mg/kg). Ten pigs were used for the macroscopic examination of the anatomy of the tonsils within the head region, and the other 10 pigs were dissected to obtain the intact tonsils that were further processed for histology and ultrastructural examinations.
Heads were cut off from 10 pigs. After the skin and mandibles were removed, the heads were sectioned sagittally into two parts. The heads were rinsed with tap water for 2 min and fixed in Bouin's fixative for anatomical examination (Zhang et al.,2007). The length and width of the tonsils were measured and pictures were taken.
Four tonsils, the soft palatine, paraepiglottic, tubal, and pharyngeal tonsils, were dissected out of each of the 10 pigs immediately after euthanasia. The samples were fixed in Bouin's fixative for 24 hr, then embedded in paraffin and 20 sections, 4 μm thick, were cut at an interval of 50 μm (Xiaowen et al.,2009). After hematoxylin-eosin staining, slides of sections were sealed with cover slips, and observed under a light microscope. The number of the lymphoid follicles was counted under a light microscope.
Tissues were first fixed in 2% glutaraldehyde for 24 hr, post-fixed in 1% osmium tetra oxide for another hour and then were embedded. Thick sections (1 μm) were first prepared and stained with toluidine blue to locate the areas of interest. Sections of 60–90 nm were prepared and mounted on metal grids. They were then stained and examined under a transmission electron microscope (Japan, JEOL, JEM-100CX II).
The Tonsil of the Soft Palate
Under visual examination, the tonsil of the soft palate could be seen as two well-circumscribed plaques in a flat oval structure lying on both sides of the median palatine raphe. The length of the tonsil varied from 18 to 20 mm and the width of a single side from 12 to 18 mm (Table 1).
Table 1. The size of the four porcine tonsils
The tonsil of the soft palate
The paraepiglottic tonsil
The pharyngeal tonsil
Under a light microscope, the stratified squamous epithelium covering the tonsil was perforated by crypts. The crypt was formed by the invagination of epithelial cells of the tonsil of the soft palate (Fig. 1a). The epithelial of the tonsil of the soft palate in porcine seem to resemble the ovine, equine, and bovine PT (Kumar and Timoney,2005a; Palmer et al.,2009; Casteleyn et al.,2010). Underlying the epithelium, a dense connective tissue layer extended into the deep parenchyma and formed many trabeculae. Both the epithelium and the underlying dense connective tissue were infiltrated by lymphoid cells. The parenchyma of the tonsil of the soft palate was mainly composed of lymphoid follicles and diffuse lymphoid tissues. The lymphoid follicles were visible at the openings of the fossules.
Transmission electron microscopy revealed that the epithelial cells of the tonsil were packed tightly, by tight junctions and desmosomes (Fig. 2a); and underneath, there was a thick layer of collagen fibers frequently infiltrated by lymphoid cells. High endothelial venules (HEVs), observed in the connective tissue underneath the epithelium (Fig. 2b), contained high endothelial cells irregularly shaped and lightly stained.
The Paraepiglottic Tonsil
By visual observation, the paraepiglottic tonsil was located laterally to the epiglottis and was a round protruding structure with a diameter varying from 7 to 12 mm (Table 1). Its structure was similar to that of the tonsil of the soft palate. By histological examination, the epithelium covering the tonsil was a stratified squamous epithelium with a thick layer of collagen fibers underneath (Fig. 1b) extending into the tonsil and dividing the parenchyma into many lobules. The lymphoid follicles were mainly distributed under the connective tissue. The number of the lymphoid follicles varied from 15 to 23, less than that in the tonsil of the soft palate. Crypts lined by the stratified squamous epithelium invaginated into the connective tissue. Some secondary lymphoid follicles distributed along the walls of these crypts. Usually, the stratified epithelium of the crypts was heavily infiltrated by lymphoid cells.
The Pharyngeal Tonsil
The pharyngeal tonsil was located at the roof of the pharynx on the caudal part of the pharyngeal septum and formed an elevation with several invaginations of the epithelium. The length of the tonsil varied from 25 to 30 mm (Table 1). The surface of this tonsil showed several folds most longitudinally oriented. Histological observation showed that the epithelium overlying the tonsil was a pseudostratified columnar epithelium frequently infiltrated by lymphoid cells (Fig. 3a). The collagen fibers were sparsely distributed underneath the epithelium. HEVs, detected more frequently than in the tonsil of soft palate and the paraepiglottic tonsil, were mainly located in the interfollicular regions. The pharyngeal tonsil was composed of scattered lymphoid follicles, aggregations of lymphoid cells, and diffuse lymphoid tissue.
Transmission electron microscopy illustrated that the epithelial cells were loosely arranged (Fig. 4a) and adjacent cells were joined by a number of desmosomes, and the microvilli size was various (Fig. 4b). HEVs in the pharyngeal tonsil were mainly composed of high endothelial cells and contain granulocytes and lymphocytes (Fig. 4c).
The TT, the smallest tonsil of the five porcine tonsils with its size varied from 6 to 10 mm (Table 1), was observed in the lateral nasopharyngeal wall, and mainly caudal, to the opening of the auditory tube. Under a light microscope, its surface was lined by a pseudostratified columnar epithelium with crypts (Fig. 3b). The epithelium was characterized by one to six rows of nuclei of various heights, and it was composed of basal cells, supporting cells, and a few goblet cells. The nuclei of basal cells were linearly arranged toward the basement membrane with their longitudinal axes parallel to the length of the epithelium. The collagen fiber layer underneath the epithelium was thin and loose compared with those in the oropharyngeal and laryngopharyngeal tonsils. The number of follicles in the parenchyma varied from 15 to 20, less than in the oropharyngeal tonsil as well. In the TT, at certain parts of the epithelium, lymphoid follicles in the parenchyma reached the basal side of the epithelium, and some lymphoid cells were even seen migrating across the epithelium (Fig. 3b).
The tonsils, which were composed of accumulation of lymphoid cells usually concentrated in lymphoid follicles, were present in the mucosa of the oropharynx, nasopharynx, and laryngopharynx (Perry and Whyte,1998). They were part of the mucosal immune system and formed a ring of lymphoid tissues at the opening of the digestive and respiratory tracts. Indeed, all parts of this ring were strategically located to perform regional immune functions because these structures were exposed to both airborne and alimentary antigens (Brandtzaeg,2003). These tonsils apparently played important immune-inductive roles as components of the mucosa-associated lymphoid tissue (Brandtzaeg,2003), since their structures also resemble those of lymph nodes, they may also function as local effector organs of the systemic-type as well as the mucosal-type of adaptive immunity (Faramarzi et al.,2006; Brandtzaeg,2011).
The tonsil of soft palate and the NT were the important lymphoid tissues of the pharynx in most domestic animals. However, the crypts themselves in pig have been implicated as portals of entry for a variety of bacterial agents including Streptococcus suis, intracellular organisms, and lymphotrophic viruses (Horter et al.,2003). Therefore, further study should be carried out to better understand the mechanism of the tonsils responding to a variety of bacterial agents.
Most of the previous studies have been undertaken mainly on the tonsil of soft palate (Williams and Rowland,1972; Williams et al.,1973; Belz and Heath,1995a,1995b,1996; Belz,1998a,1998b; Casteleyn et al.,2010), but little is known about the other tonsils (the paraepiglottic tonsil, the pharyngeal tonsil, the TT, and the LT) in the pig. Here, we studied the histological and ultrastructural features of the porcine tonsils (the paraepiglottic tonsil, the pharyngeal tonsil and the TT) to obtain a better understanding of their functions.
In this study, four porcine tonsils were categorized into two kinds: the oropharynx and laryngopharynx tonsils (the tonsil of soft palate and the paraepiglottic tonsil) and the nasopharynx tonsils (pharyngeal tonsil and the TT). The tonsils of the first type showed similar structural characteristics. The epithelia covering the tonsils were stratified squamous epithelia with a thick underlying layer of collagen fibers. However, in tonsils of the second type, the epithelia were most pseudostratified columnar epithelia with crypts. More interestingly, the epithelial cells were loosely associated, and the collagen fibers underneath were sparsely distributed. This special type of epithelial may be easy for lymphocytes to access to the mucosal surface, similar situation has been found on the ovine TT (Casteleyn et al.,2010). These findings implied that the NT may play a major role as an inductive and effective site in responding to the variety of bacterial agents. Besides, they featured more HEVs, the specialized vessels that support active lymphoid cells migration from peripheral blood to the secondary lymphoid organs (Indrasingh et al.,2002), communicating between local systemic and mucosal adaptive immunity. In conclusion, the four porcine tonsils may pay an important role in local systemic and mucosal adaptive immunity against airborne and alimentary antigens (Brandtzaeg,2003).