(A) Cell biology of epithelia

Advanced Drug Delivery Reviews, 7 (1991) 313 338

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© 1991 Elsevier Science Publishers B.V. All rights reserved. / 0169-409X/91/$03.50 ADONIS 0169409X9100340E A D R 00102

(A) Cell Biology of Epithelia Martin Mackay, Ian Williamson and John Hastewell Research Centre, Ciba-Geigy Pharmaceuticals, Horsham, West Sussex, UK (Received N o v e m b e r 15, 1990) (Accepted M a y 6, 1991)

K e y words: Epithelial cell; Epithelium; J u n c t i o n a l complex; Barrier function; Polarity

Contents Summary .........................................................................................................

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I. I n t r o d u c t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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II. Types o f epithelia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Corneal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. N a s a l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Buccal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Small intestinal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Large intestinal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. P u l m o n a r y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Vaginal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. D e r m a l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

315 316 317 318 319 322 324 325 325

III. D e v e l o p m e n t a n d differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Cell t u r n o v e r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Polarity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. J u n c t i o n a l complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

326 326 327 328

IV. G e n e r a l a s s e s s m e n t o f barrier function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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V. C o n c l u s i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: BALT, bronchus-associated lymphoid tissue; G A L T , gut-associated lymphoid tissue; G1 tract, gastrointestinal tract; M D C K , M a d i n e - D a r b y canine kidney. Correspondence: M. Mackay, Research Centre, Ciba-Geigy Pharmaceuticals, Wimblehurst Road, Horsham, West Sussex, R H I 2 4AB, UK. Fax: (44) (403) 69979.

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Acknowledgements .............................................................................................

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Summary This review aims to present the cell biology of epithelia with particular reference to their barrier function. The structure of epithelia associated with different non-parenteral and transdermal routes of drug administration is illustrated. In addition, the development and differentiation of epithelial cells with respect to cell turnover, polarity and junctional complexes are considered. I. Introduction There are over 200 cell types in the human body of which 60% are classified as epithelial. Virtually all external and internal surfaces of the human body are 'protected' by a layer of cells, the epithelium. Classically, an epithelium is a continuous sheet of cells with junctional domains between individual cells. This forms a border between different biological environments and exercises both a protective role and, more importantly with respect to drug transport, a selective permeability barrier. Epithelia consist of different specialised cell types, for example, columnar, cuboidal, polygonal, irregular, squamous and ciliated and form diverse arrangements ranging from monolayers (Fig. 1), through transitional to pseudo-stratified (distorted columnar) and stratified structures (Fig. 2). They may be protective, absorptive or secretory and are found in several locations throughout the body. Within an epithelium, many types of cell perform specialised functions (see below). Epithelia are intimately associated with many other cell types and tissues in the body and rarely function in isolation. Recent advances in molecular cell biology [1,2], state of the art techniques such as recombinant DNA methods, in vitro models [3-5], culture of polarised Heterogeneous Microvilli ~ cell types (Brush border,

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Tight junction

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~Basolateral membrane "*Basementmembrane

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Columnar epithelial cell Epithelial surface Fig. 1. Schematic representation of a m o n o l a y e r epithelium.

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Stratum Stratum Stratum gr;

space

311

Stratum

lembrane

Fig. 2. Schematic representation of a stratified epithelium.

cells on permeable supports [6-8] and transgenic technology [9] have led to a greater understanding of the structure and function of epithelial cells. The barrier properties of these cells with respect to drugs, most notably therapeutic peptides and proteins, has been known for many years. Drug transport across isolated epithelia has been investigated recently in the belief that an intimate knowledge of the pathways taken will lead to effective non-parenteral and transdermal drug delivery systems [10,11]. With respect to the theme of this special issue, the epithelial cell types associated with the different routes of drug administration considered in depth later in the issue will be elaborated upon. In addition, the properties of epithelia, development, growth considerations and an overview of the barrier function will be presented.

II. Types of epithelia This section will consider the different types of epithelia and concentrate on those associated with non-parenteral and transdermal routes of drug administration namely, corneal, nasal, buccal, intestinal, rectal, pulmonary, vaginal and skin. This account is by no means exhaustive but rather aims to highlight the differences in architecture of epithelia and the variety of epithelial cell types. Table I shows the functional types, the cell types and structure of

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TABLE I THE F U N C T I O N A L TYPES, CELL TYPES A N D S T R U C T U R E ASSOCIATED WITH THE E P I T H E L I U M OF N O N - P A R E N T E R A L A N D T R A N S D E R M A L ROUTES OF D R U G ADMINISTRATION

Route

Functional types

Cell types

Structure

Corneal

protective

stratified

Nasal

absorptive Goblet protective

stratified pseudo-

basal polygonal squamous squamous ciliated transitional

Pulmonary (i) \bronchi/ bronchioles

protective Goblet

pseudo-

(ii) Alveoli Buccal

absorptive protective

monolayer stratified

Small intestine

absorptive Goblet endocrine tuft

monolayer

ciliated cuboidal Clara squamous squamous polygonal columnar paueth

M-cells

Large intestine Vaginal Skin

absorptive Goblet protective protective

monolayer stratified trafified

columnar endocrine squamous squamous

epithelia associated with the routes of drug delivery considered later in this issue. H.1. Corneal The physiology of the eye is covered in detail in Adler's classical text [12]. The anatomy of the eye is highly organised with several tissues and complex tear fluid dynamics [13]. The cornea is the site of the eye mainly responsible for the transport of drugs by the ocular route [14]. However, drugs administered via the ocular route are also in contact with the conjunctival and nasal mucosa [15]. Although the corneal epithelium is continuous with the conjunctiva at the corneal-sclerotic junction [16], Thoft et al. [17] have shown that the characteristics of the conjunctival epithelium are markedly different from those of the corneal epithelium. During repair of corneal injury, the conjunctival epithelium transforms into corneal epithelium. Significantly, the conjunctival epithelium is thinner, less organised, possesses mucus secreting cells known as goblet cells and a good blood supply. Goblet cells are found in many epithelia [18] and will be considered in more detail later. This section will concentrate on the cell biology of the corneal epithelium and consideration of ion movement and the implication to drug transport will

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not be discussed here. The corneal epithelium of humans consists of five or six layers of cells and represents 10% of the total weight of the cornea [19]. The surface layer consists of non-keratinised overlapping squamous cells which possess microvilli, whilst the middle and deeper layers are more columnar. The microvilli increase the surface area and offer a greater area for attachment of the tear film [20]. The tear film consists of three layers namely, the superficial oily layer, 0.1 /~m; the tear fluid, ~ 7 ~tm; and the mucoid layer, ~ 0.04 ~tm. Each layer of the tear film is secreted by different cells in glands which make up the complex lacrimal apparatus of the eye. The basal layer of the epithelium consists of tightly packed columnar cells. This entire epithelium is of a uniform thickness and displays great regularity. The fine structure of the corneal epithelium has been reported by Pedler [21] and Versura and Maltarello [22] have reviewed the morphology of the eye with respect to electron microscopy techniques. The outer layers of the epithelium are relatively impermeable and function mainly to protect the lens. However, damage to the outer layers increases permeability significantly suggesting that the basal layers are less 'tight' [17]. The epithelium of the eye is highly specialised and functions mainly as a protective barrier. However, the nutrition of the cornea depends on transport of oxygen and glucose from surrounding fluids and therefore selective permeability is essential. The sensitivity and complexity of other eye tissues and functions such as tear production and drainage has significant implications when considering this site as a route for drug administration. H.2. Nasal The respiratory tract can be divided into the upper and lower airways by the junction of the larynx and trachea [23-25]. The nose is part of the upper airways which also includes the mouth, nasopharynx and larynx. The nasal cavity extends from the floor of the cranium to the roof of the mouth and from the nostrils to the upper pharynx [26]. In man, the entrance to the nasal passage has a cross-sectional area of approximately 500 mm 2 which narrows rapidly to approximately 30 mm 2 on each side [27]. The posterior nasal passage opens into the nasopharynx which has a cross-sectional area of approximately 700 mm 2. Thus it has a large surface area and most regions have a good blood supply. The epithelium varies in thickness and in degree of vascularisation and is reported to have little capacity to metabolise drugs [28]. The anterior nasal cavity is covered by a squamous and transitional epithelium and the upper cavity by an olfactory epithelium [29]. The remainder, which represents the majority of the epithelium of the nasal cavity, consists of stratified squamous cells. However, ciliated epithelia, pseudo-stratified cells with microvilli and patches of transitional epithelia also exist. Lundh et al. [30] performed a histochemical mapping of carbohydrates of the various types of epithelia in the mouse nasal cavity and found marked differences in the olfactory mucosa compared to the respiratory epithelium. It is believed that carbohydrate

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differences are of relevance in terms of microbial invasion although any significance to drug absorption is unknown. The nasal epithelium is rich in goblet cells which serve to cover the surface with mucus. In addition to the normal constituents of mucus, nasal mucus contains enzymes such as lysozyme, lactoferrin, antibodies and interferon. The combination of the ciliary beating of ciliated epithelial cells and the physical properties of the mucus secreted by serous and mucous glands lead to 'mucociliary clearance' [31]. This protective phenomenon results in the removal of dust, allergens and bacteria [32] and its arrest results in increased infections of the respiratory tract [33]. Hermens and Merkus [34] reviewed the influence of many drugs and additives on nasal ciliary movement. The list included preservatives such as chlorbutanol and thiomersal, expectorants, decongestants, antibiotics and anaesthetics. Their conclusion was that several different types of drugs affect ciliary movement and whilst acute administration may not present a clinical problem, long-term treatment for systemic effects may do. Several 'absorption enhancers' commonly used to promote absorption of peptide and protein drugs have also been shown to effect mucociliary clearance including sodium tauro-24,25-dihydrofusidate, Laureth-9, sodium deoxycholate, sodium taurocholate and sodium glycocholate [35]. In addition, the effects on the nasal epithelium of many drug absorption enhancers have been investigated [36] and it is clear that the consequences of epithelial damage during chronic administration gives cause for concern. Ennis and coworkers [36] have developed a rat nasal mucosa model and scored the effects of enhancers in terms of mucosal surface integrity, ciliary morphology, mucus/ extracellular debris and presence of red blood cells. For example, they have shown that five minute exposure to 1% sodium taurodeoxycholate resulted in severe erosion of mucosal membranes, leaving basal cells and lamina propria exposed. The surface area of the upper respiratory region is high, mainly due to the sinuses. It is believed that this area of pseudo-stratified, ciliated columnar epithelium is responsible for most drug absorption [37]. Given the large surface area, the low metabolic activity of the nasal epithelium and the generally good blood supply to the nasal tissue, it would appear that the nasal route is a strong candidate for drug absorption. McMartin and coworkers analysed the structural requirements for the absorption of drugs and macromolecules from the nasal cavity [38]. They found that although the nasal epithelial barrier performed a protective function, absorption of macromolecules can occur and concluded that molecules up to 1000 molecular weight showed good availability with a decline in availability above this value. H.3. Buccal

The buccal cavity consists of the cheek, upper and lower lips. The epithelia of this cavity is non-keratinised, stratified and consists of polygonal cells at the basal membrane leading to squamous cells at the surface. It is approximately 500--600 pm thick although this varies between different regions within the

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tissue. In the mouth, the non-keratinised tissues, such as the floor and buccal mucosa, have a greater permeability than keratinised tissue to many substances. However, the epithelium still acts as an effective rate-limiting step to absorption. Squier and Hall [39] showed that the floor of the porcine mouth was significantly more permeable to radiolabelled horseradish peroxidase than other regions such as the gingiva and buccal mucosa. The salivary pellicle is a multilayered film. It coats the buccal epithelia and acts as a protective barrier and lubricant. In addition, it retains moisture and has been shown to be a determinant of bacterial adhesion [40]. It is formed by salivary components which absorb to the buccal epithelial cells. This pellicle is thought to be unique and its function is to protect the cell from physical, chemical and osmotic damage. Squier and Lesch [41,42] studied the pathways taken by a number of compounds across this stratified squamous epithelium. They showed that the route taken was mainly paracellular and demonstrated that radiolabelled cholesterol, water, ethanol and horseradish peroxidase were absorbed via the intercellular compartment. In addition, they speculated that the intercellular material between the superficial epithelial layers is extruded by a unique organelle called a 'membrane coating granule'. Electron microscopy of the membrane coating granule in non-keratinised oral epithelia [41] revealed that cytoplasmic granules, 0.2 ~tm, appeared in the golgi region and migrated to the cell membrane where fusion occurred. Lavker [43] showed that the lamellar contents of membrane coating granules mix with existing material and form broad sheets in the intercellular spaces of rat keratinised epithelium. The sheets are oriented parallel to the cell membrane and therefore may act as a barrier to permeability. 11.4. Small intestinal Detailed descriptions of the gastrointestinal tract (GI tract) epithelia have been given by Fenoglio-Preiser et al. [44] and Johnson [45]. The epithelium of the small intestine is composed of a continuous monolayer of columnar cells and is separated from the lamina propria by a basal lamina (Fig. 2). The epithelium in the crypts of Lieberkfihn is composed of undifferentiated absorptive, goblet, endocrine, tuft and Paneth cells whereas the cells covering the villi are mainly absorptive, goblet and tuft. Endocrine cells are abundant in the small intestine and the jejunum and duodenum have many different endocrine cell types. They produce a wide range of hormones and peptides including gastrin, secretin, vasoactive intestinal peptide, substance P, bombesin, neurotensin and secretin. Endocrine cells of the small intestine can occur singly or in clusters and are small clear cells with a basal cytoplasm that contains electron-dense granules. Tuft cells, also called caveolated, brush, multivesicular or fibrovesicular cells, have a well-developed tuft of microvilli that are longer than the microvilli of the absorptive cells. Tuft cells are observed in both the crypts and on villi throughout the small intestine. The function of these cells is not known although it has been speculated that they are involved

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in chemoreception. A wide variety of functions have been attributed to Paneth cells including secretion of digestive enzymes, production of trophic factors and elimination of heavy metals [46]. There is little evidence to support this although there is little doubting their role as a secretory cell. They have abundant zinc-rich secretory granules which contain lysozyme, glycoproteins and other proteins. They display irregular microvilli and do not possess a terminal web. Small intestinal goblet cells are polarised, mucous secreting cells present throughout the epithelium with increasing frequency from the proximal jejunum to the distal ileum [47]. Moreover, goblet cell frequency is less on the villus tip than on the remainder of the villus. The apical two-thirds of goblet

Fig. 3. Light microscopy of the rat jejunum. The small intestine mucosa consists of a basal layer from which many long finger-like villi (V) project into the lumen, The lamina propria occupies the space in the villi centre and perpendicular to the villi lies a continuous sheet of muscularis mucosae (M). Magnification x 50.

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cells is distended due to the presence of many mucin granules. In addition, the apical microvilli are sparse and irregular in size and shape. The apical membrane of goblet cells is usually cup-shaped. The structure and function of intestinal mucins have been reported by Snyder and Walker [48]. Mucin is believed to protect the epithelium in three ways, namely, by creating a physical barrier, binding to luminal ions, molecules and microbes and disposal of trapped proteins.

Fig. 4. Transmission electron micrograph of a absorptive epithelial cell from a human ileum showing microvilli (M) and nucleus (N).

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The gut-associated lymphoid tissue (GALT) is often aggregated into regions known as Peyer's patches. M-cells are epithelial cells with microfolds and they overlay the lymphoid tissue. M-cells lack developed microvilli and although they contain many vesicular bodies, have a poorly developed lysosomal system. They endocytose macromolecules and transport them to intraepithelial lymphocytes. They are believed to play a major role in antigen uptake and the intestinal immune response. Several excellent reviews and original publications have been written recently on the M-cell [49-52]. The most important cells in terms of nutrient and drug transport are the absorptive cells. These cells along with other epithelial cell types are arranged in such a way as to considerably enlarge the surface area of what would be a simple cylinder (Fig. 3). This organisation consists of three levels namely, the folds (plicae or rugae) of Kerckring, villi and in the case of absorptive cells, microvilli. These specialisations result in an area of approximately 200 m z in adult humans and an increase in surface area of 50,000-fold. The microvilli, often over 1000 per cell, project into the lumen forming the so-called brush border (Fig. 4). The epithelium is clearly designed for the purpose of selective absorption of nutrients from ingested food. This specialisation does offer specific transport routes for drug absorption and this subject will be discussed later.

H.5. Large intestinal The colonic epithelium is a monolayer of columnar cells. It contains absorptive, goblet and a small number of endocrine cells. The absorptive cells possess microvilli but are not organised on a villus. However, the crypts of Lieberkiihn are long with an abundance of goblet cells [44]. Irregular folds in the intestine itself serve to increase surface area but not to the same extent as that displayed by the small intestine. Fig. 5 shows a light microscopy section of the rat colon. No villi are observed and the relatively smooth surface has irregular folds known as plicae semilunares. The crypts are clustered into small groups and extend into the muscularis mucosae. The colonic epithelium displays a regular pattern of cell differentiation from the base of the crypt to the luminal surface. The degree of differentiation is similar to that observed in the small intestine. Undifferentiated absorptive cells contain many ribosomes, a convoluted nucleus and few organelles. However, as they differentiate the rough endoplasmic reticulum develops causing the cytoplasm to become much denser. The structure of the colonic epithelium is well suited to its function of resorption of water. Although it does not possess the surface area of the small intestine the absorptive cells are arranged in a manner that enables gross water transport [45]. In addition, the mucous lubrication protects against abrasion from solid matter and enables the compaction and excretion of faeces without damage to the tissue. Towards the rectum there is a transition between the columnar epithelium of the large intestine to the keratinised stratified squamous epithelium of the anal

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skin. The anatomy of this area of transition, sometimes referred to as the anal transitional zone, is complex and the epithelium in this region highly variable [53]. The superficial cells can be basal, flattened or columnar and goblet cells and endocrine cells are present. Isolated crypts are observed and a small number of Paneth cells are also present. The mature columnar absorptive cells possess apical secretory activity reminiscent of goblet cells. The keratinised stratified epithelium of the anus contains melanin-rich cells.

Fig. 5. Light microscopy of the rat colon. The mucosa of the colon is devoid of villi and is characterised by a relatively smooth surface that is thrown into irregular folds of plicae semilunares (P). Numerous closely spaced crypts extend down into the mucosa. The lamina propria occupies the space between and beneath the crypts. Beneath the crypts and perpendicular to them lies a continuous sheet of muscularis mucosae (M). Magnification x 50.

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H.6. Pulmonary The physiology and anatomy of the lower airways has been the subject of intense investigation [30,54,55]. Weibel [55] developed a detailed anatomical model of the lung and has calculated airway lengths and diameters from a morphometric analysis. The pulmonary airways can be divided into the intrapulmonary bronchi (larger than 500 gm in diameter) and the bronchioles and the alveoli (less than 500 gm in diameter). Lung function can also be divided into three zones, namely, conducting, transitional and respiratory and the function of these zones has been discussed by Forrest [56]. The lung has an extremely large surface area ideally suited to the role of gaseous exchange. For example, Penney [57] states that there are 3-10s alveoli in humans presenting a surface of 143 m 2. Many cell types are found in the lung and these have been reviewed recently [57]. Jeffery [58] reports eight epithelial cell types of which four have been classified as secretory, namely, goblet, Clara, serous and 'special type' cells. Souma [59] has measured the distribution of epithelial cells in the rat lung using electron microscopy techniques. The bronchi are lined with a ciliated mucussecreting epithelium and these cells constitute 70-80% of epithelial cells. The epithelium is columnar and pseudo-stratified in this region and also contains goblet cells which account for approximately 25% of the number of differentiated cells in the large conducting airways [27]. However, the number of goblet cells decrease in the distal bronchioles. Moving toward the alveoli, the epithelium decrease in height and in the terminal bronchioles consists of a monolayer of cuboidal cells. In this region, ciliated cells only constitute 50% of epithelial cells. Clara cells are the most predominant type in the lower bronchioles whereas goblet cells are almost entirely absent. Although the appearance of Clara cells is consistent with that of a secretory cell the function of the cell or its secretory product is unknown. Komaromy and Tigyi [60] speculate on the main function of Clara cells being the secretion of bronchiolar lining layer components, metabolism and detoxification of xenobiotics and other toxic compounds and participation in the renewal process of the bronchiolar epithelium. However, the evidence for these functions remains tenuous. The alveolar epithelium consists of a monolayer of squamous pneumonocytes (type 1), agranular pneumonocytes (type 2) and brush alveolar cells (type 3). Type 1 cells predominate over the surface (over 95%) and display thin squamous cytoplasmic extensions. Type 2 cells are cuboidal, contain multivesicular bodies and many organelles and display blunt microvilli. They are highly metabolic. It is believed that they secrete the lungs surfactant material [61]. Type 3 cells are rarely found in humans although constitute 1% of epithelial cells in the rat. Type 3 cells may be involved in water absorption, chemoreception or endocrine function although there is little evidence to support this. M-cells also are present in the lung overlying bronchus-associated lymphoid tissue (BALT) [62]. They are found in the larger bronchi especially surrounding the bifurcations. It is assumed that pulmonary M-cells are part of the immune system and act as portals for antigenic sampling. The lung is an

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extremely complex organ. The functions of lung epithelia in protection and acting as a selective permeability barrier reflect this complexity. H.7. Vaginal The vaginal epithelium performs a protective function. In humans, it consists of stratified squamous cells of differing thicknesses. Surface cells are flattened and overlap each other whilst deeper layers are less compressed. The basal layer, for the most part, is columnar. The basal cells of vaginal epithelia are endowed with cilia. Warfvinge and Elofsson [63] found that most basal cells possessed a single cilium but were unable to assign a function to this modification. Vaginal pH is partly maintained by the sloughing off of the superficial epithelial cells into the lumen. These cells contain a high content of stored glycogen which is metabolised to lactic acid in the vaginal lumen. The pH varies with the menstrual cycle with an increase during the luteal phase [64]. The epithelial barrier is known to alter during the menstrual cycle [65]. For example, in the follicular phase, the epithelium consists of clusters of squamous cells, whilst in the luteal phase, desquamation occurs and there is an opening of the intercellular grooves. During and after ovulation, intercellular porosity increases and desmosomes dissociate leading to open intercellular connections [66]. The epithelium associated with the vagina also changes with age, pregnancy and some hormonal disorders. These changes are known to affect the absorption of drugs [66]. Given the cyclical alterations of the epithelium, it is clear that some phases will lead to increased absorption of drug whilst others offer a 'tight' barrier. The implications of this constant change in physiology to drug absorption are evident. Illum and coworkers [67] studied the vaginal absorption of gentamicin in ovariectomized rats. A morphological analysis of the epithelium was performed in rats administered gentamicin alone or with absorption enhancers 1% palmitoylcarnitine, 0.5% lysophosphatidylcholine, 1% Laureth-9 and 10% citric acid. All the adjuvants enhanced absorption but also reduced the thickness of the mucosal layer with Laureth-9 and lysophosphatidylcholine causing complete desquamation. H.8. Dermal Many reviews and texts have been published on the structure and function of the skin [68-70]. It forms the entire external surface of man and is continuous with the mucosa of the GI, respiratory and urogenital tracts, where modifications give rise to muco-cutaneous junctions. The skin is multilayered with the outermost layer, the epidermis, itself consisting of two layers; the stratum corneum or 'cornified layer' is the most superficial layer which is ten to fifteen layers of flattened, squamous, stratified, keratinised dead cells. These cells emanate from the second epidermal layer, the stratum germinativum or 'viable epidermis'. The stratum corneum varies in thickness being thickest, between 400-600

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/.tm, on friction bearing surfaces such as the palms of hands and soles of feet and thinnest, 10 ~tm, on regions such as lips, eyelids and other muco-cutaneous junctions. The stratum corneum is replenished every two weeks in adult man and this complex process is considered below. A typical keratinised epithelial cell has keratin filaments distributed amongst lipid and non-fibrous proteins throughout the cell [69]. The stratum germinativum also varies in thickness and consists of layers of cells notably, the basal layer, the spinous layer and the granular layer which underlies the stratum corneum. This is a highly metabolically active layer which undergo transitions leading to cell death (see below). Elias and Friend [71] have studied the structural basis for the permeability of mammalian skin. They showed that whilst the stratum corneum is highly impermeable to many molecules, an effective barrier also resides in the mid to upper stratum germinativum. In another study, Elias and coworkers investigated the lipid composition of the epidermal barrier and how this influenced structure [72]. They speculated that, although the intercellular space of the stratum corneum is presumed to occupy a small percentage of the volume, it is an important pathway in the permeability of non-polar molecules. Given these barrier functions, the transdermal route has proved to be extraordinarily good in terms of drug administration [73].

III. Development and differentiation III.1. Cell turnover

Fleming and Johnson have recently reviewed the early stages of epithelial cell embryological development [74]. Epithelial cells are in a constant state of regeneration. Senescent cells are perpetually lost from the epithelium, be it from the tip of villus in the GI tract or the surface of the epidermis of skin. In man, as many as 1.0.1011 cells are sloughed per day from the small intestine, which represents 40% of the cells on the villi [75]. This degree of turnover, whilst highest in the GI tract, is typical of epithelia and it is necessary to consider the proliferation and renewal of cells in the layer to understand the formation and maintenance of epithelial structure and function. For the purposes of this review, the cellular replacement of the GI tract (a simple monolayer of columnar cells) and the epidermis (a stratified epithelium of squamous cells) will be considered. The mucosa of the GI tract regenerates in the crypts found at the base of the villus. There is a band of approximately sixteen pluripotent stem cells located five cells above the base of the crypt. The cycling time for these cells is 24 h and they give rise to four transit generations of dividing cells (cell cycle, 12 h) that migrate away from the stem cell zone. Downwards migration gives rise to the Paneth cells which are located at the base of the crypt, whilst upwards migration gives rise to all the cell types found on the villus (absorptive cells, goblet cells, endocrine cells, tuft cells and M-cells) [76]. The epidermis is composed of a layer of cells, with the keratinocyte on the

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surface. The regenerative region is in the basal area of the epithelium, the stem cells accounting for 10% of the cell population. The stem cells divide with a cell cycle time of 8 days. These give rise to three generations of dividing transit cells (cell cycle time of 4 days). In total 60% of the cells in the basal region are proliferative in nature. However, it is unclear whether or not cells start to differentiate at each transit [77] or wait until all three transits have been completed [78] before undergoing terminal differentiation, to the cells found in the mature epithelial layers. It is not fully understood what causes the decline of division and the onset of differentiation as the cells migrate away from the proliferative region of the epithelia. Position is thought to play a major role in this process. Hahn and coworkers showed that in the small intestine basement membrane components such as laminin are potent promoters of cell differentiation [79]. This may be coordinated by the close spatial relationship between the mesenchymal and the epithelial cells as they comigrate towards the tip of the villus [80]. With the onset of differentiation the morphological and biochemical features that contribute to the make up the epithelia are expressed. In the GI tract differentiation of the cells continues to occur until the cells of the top third of the villus [81] are recognised as terminally differentiated displaying complete hydrolase activity [82,83] and transport capabilities [84,85]. In the epidermis the process of keratinisation begins leading to the surface keratinocyte.

111.2. Polarity The barrier or interface location of all epithelia sets constraints on their structure which manifests itself as a 'sidedness'. This sidedness or polarity is the consequence of epithelia acting as a barrier to maintain the 'milieu interieur'. Therefore, in all cases, one side of an epithelium will be the cellular environment of the body, whereas, the opposing region can range from the lumen of body cavities to the atmosphere. The skin epithelium, for instance needs to provide an externally protective surface whilst having an intimate relationship with the internal milieu of the body which provides its nutritional and structural support. However, in the case of an internal epithelium, there are less restrictions on the surface structure because of the aqueous nature of the two compartments. Therefore, these epithelia are able to interface with and in m a n y cases influence the environments they delineate. This concept of sidedness in an epithelium is observed as structural polarity. All epithelia are internally bounded by a basement membrane. In the case of a stratified epithelium there is a transverse polarity as depicted by the various layers found in the epidermal epithelium (stratum malphighii, stratum granulosum, stratum lucidum and on the surface the stratum corneum) as the cells differentiate to the terminal keratinocyte at the external pole. For a simple epithelium such as the mucosal lining of the GI tract, where a single cell layer lies above the basement m e m b r a n e , the polarity at the gross morphological level is not clear. It is not until the cellular and subcellular

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level is considered that the high degree of cellular polarisation leading to the formation of an apical pole on the surface and a basolateral pole lying at the base of the cells can be observed. In the case of the absorptive epithelium of the small intestine the hydrolase activity (e.g., sucrase isomaltase, aminopeptidase A, dipeptidyl peptidase IV, endopeptidase, phoshomonoesterase), [86,87] and transport proteins (e.g., sodium-dependent solute transport) [88,89] responsible for the final digestion and absorption of nutrients are expressed on the microvilli of the apical pole of the cells, whilst the housekeeping functions such as the sodium/potassium ATPase are located on the basolateral membrane [90] to maintain the intercellular and intracellular milieu. The maintenance of this polarity is a function of complex internal sorting of the biosynthetic pathways [1,2] and a function of the tight junctions which act as barriers to the migration of proteins and lipids of the outer leaflet of the bilayer [91] between the apical and basolateral membrane environments. In intestinal epithelium the polarity of ion transport creates a potential difference across the epithelial cell layer which can range between 10 and 50 mV, the lumen being negative [92].

111.3. Junctional complexes Many texts and reviews have been published on junctional complexes and junctional specialisations [93-95]. The properties of an epithelium result from the interactions of the cells that compose the structure and enable it to operate in a coordinated and functional manner. There are two classes of cell to cell interaction that occur. The first gives rise to the structural integrity of the epithelium, the second enables cell to cell communication. It is the sum of these two activities that account for many of the structural and functional properties of an intact epithelium. At the structural level epithelial cells are linked by contact junctions, typified in the GI tract by the tight junction and belt desmosomes (Fig. 6). These intercellular junctions connect adjacent cells into a laterally coherent sheet (see Fig. 1). The junctional complex consists of the tight junctions (zonulae occludens), intermediate junctions (belt desmosome or zonulae adherens) and spot desmosomes (maculae adherentes) [96]. The tight junction is one of the major hallmarks of absorptive and secretory epithelia [97] and contributes to the function of the transepithelial permeability barrier. The tight junction forms a continuous belt around the apical pole of the epithelial cell. Electron microscopy of the tight junction reveals the external leaflets of their opposed plasma membranes to be fused and at the internal membrane plane, linear arrays of closely packed particles form a meshwork of ridges presumed to bind the two membranes together occluding the space between [94]. In contrast to this appearance by electron microscopy, the tight junction is thought not to be a membrane fusion but consists of protein molecules which bring the plasma membranes into extremely close apposition so as to occlude the extraceUular space [91,97]. The tight junction constitutes a selective but variably efficient ionic barrier

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[92,98] sealing the intercellular space from the outside environment or extracellular space. It therefore controls the diffusion of ions and neutral molecules through the paracellular pathway, the transport route through the intercellular space [99] (see Fig. 7). Epithelia vary with respect to their ability to maintain ionic and osmotic

Fig. 6. Transmission electron micrograph of the apical region of a absorptive epithelial cell from a h u m a n ileum showing a tight junction or zonula occluden (-~), an intermediate junction or zonula adheren (A) and a spot desmosome or macula adherente (M). Terminal web (T).

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gradients between physiological solutions of different compositions. Claude and Goodenough [100] studied the freeze fracture morphology of tight junctions in several epithelia displaying a range of transepithelial resistances. They showed that 'tight' epithelia such as urinary bladder, possessed many junctional strands whereas 'leaky' and 'very leaky' such as proximal convoluted tubule, often possessed only one junctional strand. They concluded that it was possible to relate the morphology of the tight junction to transepithelial morphology. Exceptions to this have been reported and Stevenson and coworkers [101] showed that there were no differences in the cellular morphology or tight junction structure of two strains of Madin-Darby canine kidney (MDCK) epithelial cells which differ in transepithelial resistance. The tight junction is also an important contributory factor to the organisation of the transcellular pathway, the transport route through the cell (see Fig. 7). Tight junctions between cells segregate the plasma membrane into apical and basolateral domains [102,103] preventing membrane proteins and outer leaflet lipids from diffusing into the other domain. The polarisation of ion channels, pumps and enzymes into these domains is responsible for the directional nature of transepithelial transport. This feature and general aspects of epithelial tight junction biochemistry has been reviewed by Gumbiner [97]. Proximal to the tight junction is the belt desmosome. This junction serves to strengthen cell attachment in the vicinity of the tight junction as well as anchor, by means of its cytoplasmic plaque, the terminal web of the actin filaments to the plasma membrane. The electron dense intercellular space contains filaments corresponding to a calcium dependent adhesion mediating protein thought to be uvomorulin [104]. Coordination of epithelial function is dependent on cellular communication between cells. This occurs via the gap junction, which forms a transient structure between the cells and has no structural significance indicated by its temporary nature. The functioning gap junction is composed of two hexamers (one from each cell) which create a calcium-regulated aqueous pore for the passage of ions and small molecules [105]. Thus, groups of cells are formed into functionally coupled units enabling the coordination of the epithelial cell sheet. IV. General assessment of barrier function

All epithelia act in a frontier role and as such present a barrier between the environments they separate. Depending upon the function of the epithelium, the barrier may be selective (small intestine), unidirectional (absorptive), bidirectional (secretory/absorptive) or 'absolute' (skin). In general terms, the stratified epithelia present the most complete barriers to exchange between the two environments they interface. The structure of the epithelia is such that there is a multiple layer of cells (see Fig. 2) between the two environments resulting in a high degree of structural integrity which effectively blocks transepithelial transport. Epithelia which act as selective barriers tend not to be composed of

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multilayered structures and often present only a monolayer of cells between the two environments (see Fig. 1). In spite of the structural diversification of epithelia to achieve their individual barrier functions, none has managed to present the perfect barrier. All epithelia are permeable to some extent by molecules the body either wishes to totally exclude, for example toxins [106], or make unavailable across particular epithelia, for example, macromolecules in the GI tract [107]. Often the availability of these molecules is extremely small [108]; however, because such examples exist it is realistic to believe that natural pathways through epithelia can be manipulated or enhanced to the advantage of drug delivery. In order to capitalise on such pathways it is necessary to consider the barrier in more detail. To achieve this, arguments will be developed based on a theoretical epithelium and illustrated by specific examples. Fig. 7 indicates the general features of the epithelial barrier. This barrier may be the consequence of inter-organ cooperation; thus in the luminal environment of the GI tract above the epithelial layer, degradative enzymes, secreted from the pancreas present the first barrier to macromolecular absorption across the epithelia [109]. For a detailed text on the molecular and cellular basis of digestion see Desnuelle [110]. On the surface of many epithelia exists a mucus layer [111], usually secreted by the epithelial goblet cells. This acts as a extracellular barrier, that may block passage to the epithelial layer or simply bind solutes and drugs reducing their effective concentration [112]. The mucus layer may also aid the formation and maintenance of a microenvironment trapped directly above the epithelial cells. In the small intestine there is an acid microclimate [113] which can act as a barrier or enhancer to drug absorption depending on the pKa of the drug and the consequent charge state at the epithelial surface rather than in the bulk phase of the lumen as proposed by Schanker and coworkers [114]. At the o qD

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epithelial surface, a plethora of degradative enzymes may be expressed on the external surface of the cells which hydrolyse peptides, carbohydrates [87] and drugs [115]. Therefore, the first part of the barrier function to be considered when assessing an epithelium is the extracellular environment. At the epithelial cell surface there are two routes potentially available to molecules. Transepithelial delivery could be achieved either across the cell (transcellular) or through the junctions that hold the cells together (paracellular) (see Fig. 7). The transcellular route is of major importance to an absorbing epithelium which exerts the selective nature of its barrier by expressing carrier-mediated nutrient transporters [88] or receptor-mediated endocytosis [10]. These processes show a high degree of selectivity within classes of molecules, such that the pyrimidines, uracil and thymine, are absorbed to a much greater extent than cytosine [116]. Preferences are also observed in the case of sugars and amino acids [89]. The second transcellular route across epithelia relies on passive processes, by which molecules diffuse across the cell barrier. It is the physical properties of the molecule that dictate the probability of this occurring and the epithelial cell cannot exert any control over passive absorption [117]. This does not mean that one epithelial cell type may be more permeable to passive transport than another. In addition, metabolic processes within the cell may still act as an effective barrier on overall absorption, for example, the mercaptopurines [118]. To take advantage of the transcellular route it is either necessary to have drugs which are structural analogues of the naturally transported substrates such as 5-fluorouracil [119] or have the physical properties that support passive absorption [115,117]. The paracellular route takes advantage of the leakiness of the cell to cell junctions. This can vary considerably between epithelia and is often expressed in terms of integrity as measured by a physical parameter such as electrical resistance. In general, absorptive epithelia, for example the small intestine, are found to be leakier than tight epithelia such as that of the bladder [100]. The paracellular pathway is mainly open to ions and small molecules. However, the tight junctions of some epithelia are sensitive to hormonal regulation [120] and are potential sites for modification by absorption enhancers, particularly calcium chelators which open up the tight junctions of the epithelium [121]. The relative importance of transcellular versus paracellular routes will also depend on the epithelia in question. A stratified epithelium such as the skin presents multiple layers of cells and as such a transcellular route across the epithelium will be composed of many discrete and possibly additive barriers. On the other hand, the paracellular route may be continuous from one side of the epithelia to the other. As a result, when considering transport across the skin the contiguous lipidic pathway round the cells [71,72] which represents the paracellular route is more attractive than the multiple layered barrier pathway through the cells. The final consideration for the barrier function is the clearance at the base of the epithelium, this is via the vascular compartment which perfuses the base of all epithelia or in some cases may enable access to the lymphatic circulation. In

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the case o f passive diffusion across the epithelial layer, the rate o f clearance f r o m the epithelium will influence the rate o f a b s o r p t i o n . W i n n e [122,123] has given excellent a c c o u n t s o f the influence o f b l o o d flow on intestinal a b s o r p t i o n .

V. Conclusions Epithelia are highly organised cellular structures located t h r o u g h o u t the body. These structures v a r y in architecture a n d function a n d are usually intimately associated with o t h e r tissues a n d organs. T h e cells m a k i n g up an epithelium are highly specialised a n d m a n y cell types can be identified which p e r f o r m a r a n g e o f functions. Clearly the barrier function o f epithelia is extensive with respect to m a n y molecules, particularly m a c r o m o l e c u l e s such as t h e r a p e u t i c p e p t i d e s a n d p r o t e i n s . I n a d d i t i o n , the b a r r i e r m a y be c o m p l e m e n t e d by d e g r a d a t i v e processes that effectively eliminate the d r u g p r i o r to a b s o r p t i o n . This review has not given an exhaustive a c c o u n t o f the p u r p o s e o f the different cell types but r a t h e r focuses o n specific areas such as the barrier function. It is clear t h a t this function o f epithelia plays a m a j o r role in the selective t r a n s p o r t o f d r u g molecules and this will be c o v e r e d in m o r e detail during the course o f this special issue.

Acknowledgements M a n y t h a n k s to Pauline M a w h i n n e y for the p r e p a r a t i o n o f the references.

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