When is the esophageal mucosa normal?
K. Geboes (Leuven)
General organization of the esophageal mucosa
The luminal side of the normal esophagus is lined by mucosa composed of epithelium, lamina propria and a muscularis mucosae. Except for a short segment of columnar epithelium in the distal esophagus at the gastroesophageal junction, the normal esophageal epithelium is a tough, nonkeratinizing, stratified, squamous epithelium. The lower border of the squamous epithelium is irregular due to the presence of transitory folds of the lamina propria and more particularly, due to the presence of high conical papillae of connective tissue. These papillae are highly vascularized. On routine microscopy the squamous epithelium can be divided into basal, intermediate or prickle cell layers and superficial layers. In the superficial layers, functional and surface layers can be distinguished. The latter lay adjacent to the lumen. In many articles authors refer also to a basal zone. This is the area composed of the basal cell layer and several layers of cuboidal basophilic cells immediately above (the suprabasal cell layers). The upper extent of the basal zone has been arbitrarily defined as the level where the nuclei are separated by a distance equal to their diameter [1-3]. The parabasal cell layer or compartment, is roughly similar to the area classically described as intermediate or prickle cell layer.
Several parameters have been introduced to assess this organization. These include total epithelial thickness, papillary height and basal zone thickness. In morphometric studies, the values of these parameters have been determined in comparison with the total thickness of the epithelium. The basal zone normally comprises no more than 15-20% of the total epithelial thickness, while the normal value for papillary height ranges from 50%-75% [4-7] (Fig. 1). There is some variability in the criteria used in different studies. The organization and relative size of the different compartments of the squamous epithelium is generally the same throughout the whole length of the esophagus. Only small regional differences have been noted. From studies in normal control individuals it appears that the relative height of the papillae and of the basal zone may be greater in the distal (2-5 cm) esophagus. Weinstein  showed that the basal zone and papillary height can even be increased in an area between 2.5 and 10 cm of the distal esophagus in asymptomatic subjects. Yet, abnormal reflux was not excluded in these asymptomatic controls by objective methods. A low false positive rate of basal zone widening and increase of papillary height was found in a study on 18 asymptomatic volunteers in whom pH reflux tests were performed to exclude pathologic reflux .
In the early morphometric studies the histologic data have been determined on routinely hematoxylin and eosin (HE) or periodic acid schiff (PAS) stained, well oriented (suction) biopsies. The PAS stain was used to show the presence or absence
Figure 1. .Normal histology of the human esophagus showing the squamous surface epithelium with lower irregular border due to the presence of high conical lamina propria papillae. Papillary length and total epithelial thickness can be used for the evaluation of the histologic picture (HE x40).
of glycogen. Cells of the basal cell layer lack glycogen. Accumulation of cytoplasmic glycogen starts above the basal zone and is considered a marker of maturation.
Routine histology and cell renewal
The squamous esophageal epithelium is a dynamic cell population which is renewed continuously. In health and in benign inflammatory conditions the proliferative indices remain in a steady state so that the cells produced are equivalent to those lost.
The different cell layers described in the squamous epithelium are the morphological expression of processes of proliferation, differentiation or maturation and dying cells. This was already realized when the basal zone was identified and its existence was explained by the presence of immature cells.
The proliferative compartment of the squamous epithelium resides in the basal cell layer. The basal or germinative layer is made up of one layer of cylindrical, basophilic cells resting on a basement membrane. The longitudinal axis of the cylindrical basal cells is oriented perpendicular to the basement membrane. Random mitotic figures can be observed by routine microscopy in this layer. Immunohisto-chemistry using antibodies directed against bromodeoxyuridine (BRDU), a compound which is incorporated in the nucleus during DNA synthesis, demonstrates the presence of S-phase cells in the same layer (Fig. 2). These cells are randomly
Figure 2. .Normal esophageal mucosa: immunohistochemistry using antibodies directed against BRDU, showing the presence of cells with positively staining nuclei in the basal cell layer (immunoperoxidase x200).
distributed. For the normal esophagus, the labeling index of the basal cell layer indicating the ratio between the number of cells showing a positive nuclear staining for antibodies directed against BRDU and the sum of the labeled and unlabeled cells is 8.7%. For the entire mucosa the labeling index is 0.95% (pulse labeling). This indicates a slow rate of cell renewal . The tissue turnover time (time taken for an entire epithelium to be regenerated) has been estimated to be approximately 2 weeks. The exact quantification of esophageal epithelial cell renewal and the most sensitive method to do so have, however, not yet been established. Various other in vitro and in vivo methods are available and have been used in different clinical conditions such as normal squamous mucosa and Barrett's esophagus. Cell proliferation studies with markers such as Ki67 (a nuclear antigen which is expressed in all phases of the cell cycle except Go) in squamous mucosa from patients with Barrett's esophagus show a mean percent of positive cells of 14.14 (± SD 12.70) . Studies have also been performed with proliferating nuclear antigen (an auxiliary protein for DNA synthetase) and this topic is still in evolution . Some basic concepts have already been established in animal studies. It appears that in rats virtually every cell of the basal layer becomes labeled after repeated injections with tritiated thymidine which indicates that all basal cells are capable of desoxyribonucleic acid synthesis and presumably of cell proliferation. One, both or neither of the two daughter cells of any mitosis can be transferred to the upper differentiating layers.
Cell proliferation and maturation is influenced by epidermal growth factor (EGF),
a mitogenic polypeptide which in man is immunohistochemically demonstrable in the subepithelial capillaries . Certain molecules, such as tumor necrosis factor and transforming growth factor ß, can inhibit growth in the esophageal epithelium under normal circumstances. Both these factors are constitutively expressed in the human basal normal esophageal epithelium .
Epithelial cell layers
The basal cell layer can be divided in two topographically different but contiguous compartments, both forming the junction with the lamina propria connective tissue. One compartment is the flat layer laying parallel with the longitudinal axis of the esophagus. The second is lining the stromal lamina propria papillae and lying perpendicular to the luminal axis of the esophagus. These two compartments show some differences in cell properties as indicated by immunohistochemical studies on the presence of cytokeratins. Cytokeratin 14 is expressed diffusely in the cells of both compartments of the basal layer, whereas cytokeratin 19 is mainly expressed in the basal cells lining the stromal papillae .
As the cells leave the germinal layer, they disconnect from the basal lamina and become larger, polyhedral and subsequently more flattened and constitute the multilayered prickle cells. The deeply situated cells are the more immature cells (suprabasal layers). The cells mature as they migrate towards the surface. As indicated earlier, immaturity can be demonstrated by PAS but more recently immunohistochemical studies have been developed using other markers. In the esophagus the pairs of cytokeratins vary in the different compartments with the degree of differentiation of the epithelial cells. Keratin types 14 and 19 have been demonstrated in the cells of the basal compartment (basal cell layer and suprabasal cell layers) and types 1 and 10 have been found in the parabasal cells. A decreased expression of Lewis blood group antigens (markers of squamous maturity) has been shown in the parabasal cell compartment. Epidermal growth factor receptor (EGFR) is expressed in the basal and immediately suprabasal epithelial cell layers where it appears as a peripheral membranous staining and is absent from the more superficial layers. EGFR is an integral plasma membrane glycoprotein for which EGF is the ligand. In normal esophageal mucosa the proportion of epithelium staining positively for EGFR has been estimated to be 29% of the total (which is almost double the value normally accepted for the basal zone area using routine HE staining or PAS staining) (Fig. 3). The staining distribution for EGFR is the same at all levels of the esophagus. The immunohistochemical studies thus confirm the existence of a basal zone composed of immature cells but the normal quantitative values obtained with these techniques are not exactly the same as those assessed with more routine stain-ings . Depending on the markers used, there may be transition zones between the basal cell layer, the suprabasal layers, the parabasal or intermediate layers and the superficial layers.
Above the basal zone the postmitotic epithelial cells mature. This process is characterized by nuclear pyknosis, loss of glycogen, loss of maturation markers and
Figure 3. .Normal esophageal mucosa: immunohistochemistry using antibodies directed against Epidermal Growth Factor Receptor showing positive membranous staining of the lower cell layers in the basal zone (BZ) and along the stromal papillae * (x40; cryostat section).
a change in cell polarity from vertical to horizontal. The cells become more elliptical and increasing amounts of tonofilaments (keratins) and desmosomes can be observed with transmission electron microscopy (TEM) . In situ hybridization allows identification of cytokeratin 4 mRNA expression starting in this (parabasal) area (Fig. 4).
As epithelial cells mature they move further away from the capillaries and oxygen. Cells in the outermost 4-6 layers accumulate lipid droplets with subsequent damage to cell organelles and intercellular junctions begin to loosen. Cell degeneration occurs in the surface layers. Superficial esophageal epithelial cells occasionally show features of programmed cell death characterized by the occurrence of apoptotic bodies but most cells seem to be lost by desquamation or exfoliation. This may be the result of lysis of desmosomes by endogenous proteinases.
Immunohistochemical studies can be used to identify markers associated with cell maturation but they can also identify other markers associated with cell activation or metabolism. Major histocompatibility class (MHC) antigens can be considered to some extent as activation markers. MHC class I antigens are normally expressed on the membrane of all nucleated cells and they appear also on esophageal epithelial cells. MHC class II antigens are mainly expressed on immunocompetent cells. Their presence has been associated with antigen processing and presentation and with cellular interactions. Increased or aberrant expression can be observed in inflammatory conditions and depends upon the release of cytokines. In the normal esophageal epithelium MHC class II antigens are expressed on a minority of epithelial cells.
Figure 4. .Normal esophageal mucosa: localization of cytokeratin 4 mRNA in esophageal epithelium by nonradioactive in situ hybridization. Positivity starts in the parabasal compartment and extends into the lower superficial layers (provided by A. Viaene and J. Baert, IRC, KUL-Kortrijk; original magnification x20).
Specialized cells in the epithelium
A variety of cell types such as neuroendocrine cells (Merkel cells), rare melanocytes, lymphocytes and Langerhans cells are normally present within the squamous epithelium of the esophagus. Lymphocytes and Langerhans cells play a major role in the immunologic defense system of the esophagus. The Langerhans cells act as antigen-presenting cells. The interepithelial lymphocytes mainly belong to the suppressor/cytotoxic phenotype (CD8). On morphology they appear as small round cells with hyperchromatic nuclei. They show an uneven distribution within the epithelium with a higher number in certain areas which appear to be these overlying lamina propria lymphoid follicles [16,17]. The presence of small round lymphocytes and Langerhans cells within the esophageal epithelium can therefore not be considered as a sign of pathology. The appearance of wiggly cells or lymphocytes with squiggly nuclei is probably abnormal . Occasionally mast cells and single eosinophils can be found in the epithelium. The meaning of this finding is not clear. According to some authors this would not be a sign of pathology in adults, but in children the presence of even single eosinophils would correlate with abnormal reflux.
Neutrophils are normally not present in biopsies from adults but might occur in small numbers in children. In fact this means that the upper limits of what is normal, or the lower limits of abnormality have not yet been determined, certainly not for the number of inflammatory cells infiltrating the squamous epithelium.
Lamina propria and muscularis mucosae
The lamina propria rests on a two-layered muscularis mucosae. The lamina propria contains lymphatics, blood vessels, nerve fibers and occasional inflammatory cells. Eosinophils can be observed in 10-33% of asymptomatic control patients. Lymphocytes can occur as scattered solitary cells or be organized in lymphoid follicles. The follicles may be associated with the excretory ducts of esophageal glands.
The value of the criteria that can be used to determine whether the esophageal mucosa is normal or abnormal depends upon the methods of investigation. The criteria established with routine microscopy may not be exact quantitatively, and routine staining may not be the most sensitive method to assess them, but in general they correlate rather well with the findings on cell maturation and cell renewal obtained by more sophisticated methods and hence with the physiology of the esophageal mucosa.
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