Squamous cell metaplasia in the human lung: molecular ... · Squamous cell metaplasia in the human...

Virchows Archiv B Cell Pathol (1991) 61:227-253 �9 Springer-Verlag 1991

Squamous cell metaplasia in the human lung: molecular characteristics of epithelial stratification Rudolf E. Leube 1 and Todd J. Rustad x

Institute of Cell and Tumor Biology, German Cancer Research Center, lm Neuenheimer Feld 280, D-6900 Heidelberg, Federal Republic of Germany

Received July 5 / Accepted July 15, 1991

Summary. Squamous cell metaplasia (SCM) is a frequent epithelial al terat ion o f the h u m a n t racheobronchia l mucosa. This review pays part icular a t tent ion to the fact that SCM can mimic esophageal, and in some instances even skin-type differentiation, showing striking similarities not only in m o r p h o l o g y but also in terms o f gene expression. Therefore , character izat ion o f this dynamic process lends insight into the process o f stratification, squamous cell format ion , and "ke ra t i n i za t i on" in a pathological ly relevant in vivo situation in man.

First, the concept o f metaplasia is presented with certain historical viewpoints on histogenesis. Then, the morphologica l characteristics o f normal bronchial epithelium are compared with the altered phenotype o f cells in SCM. These changes are described as a dis turbance o f the finely tuned balance o f differentiation and proliferation th rough the act ion o f a variety o f extrinsic and intrinsic factors. Molecular aspects o f altered cell/cell and cell/extracellular matrix interactions in stratified compared with single-layered epithelia are discussed with reference to SCM in the lung. Intracellular organi- zat ional and composi t ional changes are then summarized with special emphasis on the differential distribution o f the cytokera t in (CK) polypeptides. Finally, the still unresolved problems o f the histogenetic relationships between normal bronchial mucosa , SCM, and pul- m o n a r y neoplasms are addressed. As these quest ions remain open, examples for detection o f well defined '" marke r s " are provided that may be employed as objec- tive criteria for determining clinically impor tan t cellular differentiation features.

Key words: Metaplasia Lung D e s m o s o m e - C y t o k e r - atin Ki-67

Materials and methods

Tissue samples. Small portions of human bronchus were obtained from fresh surgical specimens (generously provided by Dr. K.

Offprint requests to: R.E. Leube

Kayser, Clinic for Thoracic Medicine, Heidelberg, FRG), snap- frozen in liquid nitrogen within 2 h after resection and stored at - 80 ~ C until used. Seven of the 23 cases examined contained suitable, often multiple squamous metaplastic loci, and were included in this study.

Cells from bronchial washings, obtained during bronchoscopy (generously provided by Dr. H.-G. Manke, Clinic for Thoracic Medicine, Heidelberg, FRG), were pelleted, washed with phos- phate-buffered saline and spread onto glass slides. Immediately afterwards, cells were fixed for 5 min at - 20 ~ C in methanol and for 10--20 sec in acetone at - 2 0 ~ C, air-dried and stored at -20* C until used.

Immunofluorescence microscopy. Tissue sections (5 Ilm thick) were prepared at -25* C, and best results were obtained when sections were allowed to air-dry overnight prior to fixation with acetone for 10 min at -20* C. Sections, serial to those used for immunohis- tochemistry, were stained with hematoxylin and eosin for histological evaluation.

The following primary monoclonal antibodies were used for the epifluorescence micrographs shown in this paper.

I) Antibody Ks8.17.2, specific for CK 8 (Progen Biotechnics, Hei- delberg, FRG). 2) Antibody M20, specific for CK 8 (Van Muijen et al. 1987; kindly provided by Dr. G. Van Muijen, Leiden, University of Leiden, Netherlands). 3) Antibody Ksl8.174.1, directed against CK 18 (Moll et al. 1988; Progen Biotechnics). 4) Antibody 6B10, reactive with CK 4 (Van Muijen et al. 1986; kindly provided by Dr. G. Van Muijen). 5) Antibody 1C7, specific for CK 13 (Van Muijen et al. 1986; kindly provided by Dr. G. Van Muijen). 6) Antibody Ksl3.1, recognizing CK 13 (Moll et al. 1988; Progen Biotechnics). 7) Antibody AEI4, recognizing CK 5 and certain hair shaft proteins (Lynch et al. 1986; kindly provided by Dr. T.-T. Sun, New York University, School of Medicine, New York, NY, USA). 8) Antibody E3, reacting with CK 17 (Guelstein et al. 1988; kindly supplied by Dr. S. Troyanovsky, All-Union Cancer Research Center, Moscow, USSR). 9) Antibody Kk8.60, specific for CKs 10/11 (Huszar ct al. 1986; Bio-Makor, Rehovot, Israel). 10) Antibody lu-5, reactive with various type I and type II CKs (Franke et al. 1987a; Boehringer, Mannheim, FRG). 11) Antibody Vim-3B4, reacting with vimentin (Herrmann et al. 1989; Boheringer, Progen Biotechnics). 12) Antibodies DPI/II, reacting with desmoplakins I and II (Cowin et al. 1985; Progen Biotechnics).

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13) Antibody DG 3.10, specific for desmoglein (Schmelz etal. 1986). 14) Antibody HD-233, recognizing the 230 kDa bullous pemphigoid antigen (Owaribe et al. 1991). 15) Antibody Ki-67, recognizing a nuclear antigen which is expressed in proliferating cells (Gerdes et al. 1983, 1991; Dianova, Hamburg, FRG).

In addition, affinity purified antibodies (gp 15.1) from a guinea pig that had been immunized several times with a purified recombinant E. coli fusion protein consisting of a part of protein A and the carboxyterminal tail domain of human CK 15 were used (for details see Heid, Bartek, Leube, Moll, Kaufmann and Franke, in preparation). Broad-reactive CK antibodies raised in guinea pig (gp 10) have been described previously (Franke et al. 1978).

Texas Red-labeled and fluorescein-isothiocyanate-coupled species-specific secondary antibodies (Dianova, Hamburg, FRG) were used for indirect immunofluorescence.

General features of metaplasias

Metaplasias are defined in standard textbooks as the transformation of a given differentiated tissue into a differentiated tissue of another, related type. The resulting temporally and/or spatially inappropriate presence of certain tissue components is the consequence of pro- found alterations in gene regulation caused by chronic irritation. Metaplasias are reversible lesions that occur as adaptive changes in a wide variety of tissues and have been regarded by some researchers as precursor lesions of malignant tumors (for review see Beresford 1981; Lugo and Putong 1984; Slack 1986).

Several hypotheses have been put forward to explain the histogenetic mechanisms of metaplasia. Rudolf Vir- chow, who was the first to present a coherent concept of metaplasia, proposed that cells can transit directly from one differentiated state to another which he described as "Persistenz der Zellen bei Ver~inderung des Gewebscharakters", and which he believed to be the underlying principle of developmental differentiation and tissue plasticity in the adult (Virchow 1871, 1884). Soon after an alternative scheme of indirect metaplasia was proposed (Lubarsch 1906; Schridde 1907, 1909). Thus, differentiated cells first de-differentiate into more immature, "intermediate" cells (phase of neoplasia). These immature cells have an increased proliferative potential and may, under certain conditions, follow an alternative pathway of differentiation (phase of metaplasia). Increased susceptibility and responsiveness to extra- neous stimuli, in particular during mitosis, appear to be precipitating factors in both phases. The notion of metaplasia as an aberrant proliferative process and the recognition of regenerative stem cells usually located in the basal cell layer of epithelia were the basis for the model that links the inappropriate tissue formation to an inappropriate differentiation of altered stem cells (stem cell metaplasia; Krompecher 1905; Teutschlaender 1919). At the same time it was proposed that dispersed pluripotent cells or quiescent differentiated cells are acti- vated and continue their intrinsic developmental program (prosoplasia; Schridde 1907, 1909; Teutschlaender 1919).

Metaplasias have been observed in almost all tissues and body sites (for review see Beresford 1981; Slack

1986). Among epithelial tissues, the most pronounced and common alterations include squamous cell metaplasias (SCM) and mucous cell metaplasias. Virtually all complex epithelia, i.e. those with a rather flat basal cell layer and columnar-like, luminal cells with specialized secretory, absorptive or transport functions, are able to form SCM in vivo. Therefore, in man, SCM have been described in glandular epithelia of the breast, prostate and mouth (e.g., Anzano et al. 1980; Fisher et al. 1983; Dardick et al. 1985; Reddick et al. 1985; Eyden and Wil- liams 1988), in epithelia of the male and female urogenital tract (e.g., A1 Adnani 1985; Gigi-Leitner et al. 1986; Fukushima et al. 1987; Weikel et al. 1987; Levy et al. 1988) and, most notably, in the epithelial lining of the air conducting system (e.g., Auerbach et al. 1957, 1961 ; Niimi et al. 1987; Yamamoto et al. 1987; for further references see below). The different frequencies of the lesions observed in these tissues seem to be related to differences in the accessibility of irritants to the target cells. The induced changes are reversible and may spontaneously recede after removal of the irritant (for lung, see Auerbach et al. 1962). Furthermore, the reverse phe- nomenon, namely mucous cell metaplasia, has been described in stratified epithelia such as the esophagus (Bar- rett esophagus; e.g., Spechler and Goyal 1986; Paull et al. 1976) or the epidermis under certain experimental conditions (Sweeny and Hardy 1976; Elias and Friend 1976).

The ability of any given tissue to undergo SCM is not only independant of malignant transformation (e.g., Bogomoletz 1982; Fisher etal. 1983; Moyana 1987; Oberman 1987; Lifschitz-Mercer et al. 1988) but also serves as the major criterion in the differential diagnosis of squamous cell carcinomas arising from complex epithelia such as the bronchial epithelium (e.g., McDowell et al. 1978b, 1980; Blobel et al. 1984; Said et al. 1983b; Broers et al. 1988).

Morphology of normal bronchial epithelium and squamous cell metaplasias in human

The characterization of normal-appearing bronchial mucosa is a prerequisite for understanding SCM. The normal tracheobronchial epithelium is organized in a polar fashion, as the basal plasma membrane of all cells is in contact with the basal lamina. However, not all cells reach the lumen and two or more rows of nuclei erron- eously suggest a multilayered composition. To stress the compositional complexity the term complex epithelium will be used.

The more basally located cells are generally considered to be undifferentiated reserve or stem cells (McDowell et al. 1978a; Inayama et al. 1988). These cells share a large surface area with the very thick basal lamina to which they are attached by numerous hemidesmosomes (Kawanami et al. 1979; Tandler et al. 1981). The dark cytoplasm contains few organelles and some tonofilaments (McDowell et al. 1978 a). Neuroendocrine cells are sparsely distributed throughout the basal cell compartment and form neuroendocrine bodies only in

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certain locations. These cells have rather dark nuclei surrounded by a pale cytoplasm containing abundant dense-core granules and probably clear-appearing neuroendocrine storage vesicles as well (Bensch et al. 1965; McDowell et al. 1976, 1978a, 1980; Gould et al. 1983; Lee et al. 1987).

All adluminal cells are of the high columnar type. The slender processes of these cells seem not to be attached to the basal lamina by hemidesmosomes (Kawan- ami et al. 1979). Therefore, tissue coherence and stability may be mediated by contacts to neighboring luminal and basal cells (Evans and Plopper 1988). Apicaily, luminal cells seal the epithelium through junctional com- plexes consisting of the belt-like zonulae occludentes and adhaerentes as well as the spot-like desmosomes and con- nexons below. As seen in the electron microscope, tonofilaments are far more sparse than in basal cells. Among adluminal ceils one can differentiate those with abundant ciliae from those with mucous droplets and apical microvilli, and others with a reduced degree of specialization that are either mucous-secreting cells in a post- release-state or immature cells with the ability to differentiate into either ciliated or goblet cells (Rhodin 1966; McDowell et al. 1978a, 1980).

Biopsies of human bronchi and post mortem examina- tions often reveal a multitude of deviations from this prototypic composition such as substantial loss of ciliated cells, increase in mucous-secreting cells (mucous cell hyperplasia) and proliferation of basal cells (basal cell hyperplasia). The most striking alteration, however, is SCM which frequently occurs at the bifurcations of larger and smaller bronchi (Niskanen et al. 1949; Valen- tine 1957). These plaque-like regions consist of stacks of flat cells filled with abundant tonofilaments (Lindberg 1935; Wittekind and Striider 1953a; Auerbach etal. 1957; Nasieil 1963, 1966; Trump et al. 1978; McDowell et al. 1980; Yamamoto et al. 1987). Loss of contact with the basal lamina and acquisition of a squamoid, non- polarized phenotype are the most prominent biological characteristics of SCM. Therefore, suprabasal cells are completely surrounded by numerous large desmosomes that form the characteristic intercellular bridges of a stratum spinosum-like cell layer (Trump et al. 1978). In some instances, parakeratosis may occur (Valentine 1957; Nasiell 1966) and epithelium and underlying mesenchyme sometimes interdigitate to form rete ridge-like structures (Askanazy 1919; Wittekind and StriJder 1953a; Valentine 1957; Nasiell 1963). SCM often occurs without any transitional zone from normal bronchial mucosa but occasional tongue-shaped structures are formed by SCM at the boundaries where they seem to taper beneath the normal epithelium which is separated from the metaplastic cells by large intercellular spaces (Nasiell 1963). Th detailed and instructive descriptions of the ultrastructural changes associated with SCM induced in animals (e.g., Wong and Buck 1971; Gould et al. 1971 ; Harris et al. 1971 ; Klein-Szanto et al. 1980b; Chopra 1982) differ in some respects, such as the degree of cellular desquamation and are, therefore, only partially applicable to human SCM.

To date, no agreement has been reached on the deter-

mining sequence of events leading to SCM in man. Loss of ciliated cells and concurrent mucous cell hyperplasia are considered to be very early changes (Black and Ack- erman 1952; Trump et al. 1978; McDowell et al. 1980; Niimi et al. 1987). Basal cell hyperplasia, another possible precursor of SCM, is characterized by the occurrence of several layers of elongated basal cells with plump, pale nuclei showing high mitotic rates, often in association with round cell (lymphocytic) infiltration (Auerbach et al. 1957; Valentine 1957; Auerbach et al. 1961 ; Nasiell 1963; Niimi et al. 1987). These proliferating basal cells push ciliated or mucus-producing cells upwards; these cells will gradually flatten, lose ocntact with the basal lamina and slough off (Nasiell 1963; Niimi et al. 1987; Yamamoto et al. 1987), a process reminiscent of the shedding of attenuated adluminal columnar cells during development of the esophagus and epidermis. Other transient stages presumed to be precursors of SCM have been described as "transitional" (Weller 1953; Valentine 1957; Nasiell 1963, 1966) or "stratified" (Auerbach et al. 1957; Niimi et al. 1987). There is no conclusive evidence that the epithelium in these latter phenotypic alterations is truely stratified, as cells may still be in contact with the basal lamina through slender miniscule extensions that are not visible in the light microscope.

The marked decrease in the number of epithelial alterations in exsmokers emphasizes the reversibility of SCM in human. The conspicuous detection of contracted nuclei surrounded by basal cells with enlarged vesicular nuclei and columnar cells with disintegrating nuclei suggest that cell death is associated with this type of tissue renewal (Auerbach et al. 1962).

Extrinsic factors influencing the development of squamous cell metaplasias

A large number of chemical agents and physical conditions can induce SCM in the bronchial mucosa. These stimuli act either alone or in combination on the target cells that remain unidentified. Although their mecha- nism of action is unknown, it undoubtedly interfers with cellular differentiation and proliferation. This section fo- cuses on a few of these extrinsic inducers of SCM.

Thus, SCM may result from, or accompany inflammation, since it is common in patients suffering from chronic lung conditions such as mucoviscidosis, bronchopulmonary dysplasia or bronchiectasis (Black and Ackerman 1952; Bonikos et al. 1976; Mithal and Emery 1976). In most examples of SCM, many inflammatory cells can be seen which may release factors that could act as mitogens and/or modifiers of differentiation on the surrounding epithelium. Accordingly, McDowell and coworkers (1990) observed an increased labeling of presumed precursor cells of SCM by bromodeoxyuridine in foci of inflammation in the hamster.

SCM has been found in association with viral infection. Thus, it was frequently diagnosed during the big postwar influenza pandemia (Askanazy 1919) and can be induced by viral infection in animals (Richter 1970). A recent report demonstrates the presence of human papillomavirus in SCM (B6jui-Thivolet et al. 1990).

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However, the most common irritant causing SCM in man is cigarette smoke and there is a well-established dependence between the extent and type of SCM and the number of cigarettes smoked (e.g., Auerbach et al. 1957, 1961; Math6 et al. 1986). Therefore, the tissue samples used for this study were all taken from heavy smokers. Cigarette smoke contains carcinogens and other epithelial irritants and induces chronic bronchitis.

Controlled studies in animals, certain transplant systems and refined cell culture systems directly support the propensity of a number of defined chemical compounds to induce SCM in the bronchial epithelium. Most of these substances are well-known carcinogens such as benzo(a)pyrene-ferric oxide (Harris et al. 1971; Schreiber et al. 1974; Stenb~ick 1973; Becci et al. 1978a, b; Barrett et al. 1980), 7,12-dimethylbenzo(a)anthracene (Niskanen 1949; Marchok etal. 1977; Klein-Szanto etal. 1980a, b, c, 1981, 1986), 3-methylcholanthrene (Gould et al. 1971 ; Cone and Nettesheim 1973), diethylnitrosamine (Reznik-Sch/iller 1980), certain chromium compounds (Levy and Venitt 1986) or asbestos (Moss- man et al. 1980, 1984; Woodworth et al. 1983; Cameron et al. 1989; Marsh and Mossman 1991), all of which may act by direct contact on the bronchial epithelium. Similarly, inhaled gases such as formaldehyde vapors (Maronpot et al. 1986; Monticello et al. 1989; Ura et al. 1989) or sulfur dioxide (Asmundsson et al. 1973) may result in SCM. These factors can act in concert thereby potentiating the risk of SCM formation and consequent malignant transformation (e.g., Mossman et al. 1984; Keenan et al. 1989).

Mechanical injury can also induce SCM in animals (Gould et al. 1971; Keenan et al. 1982a, b, c, d) and intratracheal intubation or tracheostomy can lead to SCM in human (Trump et al. 1978). Physical disruption of epithelial integrity may thereby expose responsive target cells to environmental stimuli.

Differentiation and proliferation in squamous cell metaplasias

The above mentioned extrinsic factors disturb the balance of growth and specialization in bronchial epithelial cells. The yin-yang relationship of differentiation and proliferation is a well known principle that regulates renewal and remodeling of any body tissue in a highly complex manner. Vectorial organization of stratified epithelia is presumably a reflection of increased specialization and reduced growth capacity in upper cell layers (for a recent comprehensive review concerning epidermis, see Fuchs 1990). In skin, stem cells are distributed throughout the basal cell layer. These cells have an extremely slow cycling time, thereby retaining 3H-thymidine for very long periods of time ("label retaining cells") and they exhibit a tightly regulated balance of self renewal and differentiation into cells with a very high mitotic rate (Cotsarelis et al. 1989). These latter cells compose the transient amplifying compartment that is only in part located in the basal layer but also includes cells in the first suprabasal cell layers (Lavker and Sun 1983).

After several cycles of cell division (probably between three and five) the cells start with terminal differentiation which is, however, not directly linked to mitotic arrest (Leigh et al. 1985).

One of the most widely applied proliferation markers is monoclonal antibody Ki-67 whose cell cycle regulated nuclear antigen has been partially sequenced (Gerdes et al. 1983, 1991). In patients with the hyperproliferative skin disease psoriasis vulgaris, epidermal keratinocytes of the transient amplifying compartment react with Ki- 67 (Beurskens et al. 1989). We applied this antibody to bronchial epithelia including SCM and compared the resulting immunofluorescent staining with that obtained after reaction with fetal and adult esophagus (Fig. 1). Only very few cells react with Ki-67 in normal adult epithelia wheras increased antibody reactivity is seen in SCM, as well as in hyperproliferative bronchial mucosa and fetal epithelia, indicating elevated cellular growth rates in these tissues. These observations are in agreement with the notion that experimentally induced SMC is not only a consequence of cell proliferation but also exhibits elevated growth rates (e.g., Marchok et al. 1975, 1977; Chopra 1982; Nettesheim etal. 1982; Keenan et al. 1982a, b, c, 1983; Mossman et al. 1984; Cameron etal. 1989; Niles etal. 1990; Marsh and Mossman 1991). This high proliferative activity, however, seems to be strictly limited to the area of stratification and is contrasted by low proliferative activity in the adjoin- ing mucosa (Harris et al. 1973; Chopra 1982; McDowell etal. 1984a; Strum et al. 1985). Furthermore, Ki-67 reacted predominantly with suprabasal cells and only occasionally with nuclei of basal cells in SCM (Fig. 1). These Ki-67-positive cells may correspond to the cells of the transient amplifying compartment identified in skin, presumably giving rise to suprabasal squamous cells. 3H-thymidine- and bromodeoxyuridine-labeling studies, in addition to colchicine arrest experiments, are in close agreement with this interpretation as they iden- tify dividing non-ciliated cells in basal and intermediate positions albeit at somewhat differing percentages depending on the animal and model system used (e.g., Kaufman et al. 1972; Harris et al. 1973; Marchok et al. 1975; Barrett etal. 1976; Klein-Szanto etal. 1980a; McDowell et al. 1980; Chopra 1982; McDowell et al. 1984 a; Mossman et al. 1984; Chopra and Cooney 1985; Strum et al. 1985; Evans et al. 1986; Obara et al. 1988; Rutten et al. 1988 a).

The balance between differentiation and growth is influenced by soluble extracellular factors most of which act on specific membrane-bound receptors which in turn switch on a cascade of intracellular processes using various signaling mechanisms (for review in lung, see Kelley et al. 1990). A lot of attention has been given to the impact of TGF (transforming growth factor)/3 polypeptides on bronchial epithelial cells. These factors promote squamous differentiation and inhibit growth, thereby also affecting c-myc proto-oncogene expression and DNA methylation (Jetten et al. 1986; Masui et al. 1986; Wilson et al. 1988; Pfeifer et al. 1989; Ke et al. 1990). High concentrations of biological active TGF /3 have been extracted from the epithelial lining fluid of the

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lower respiratory tract in man that could be secreted by resident alveolar macrophages (Yamauchi et al. 1988) and/or epithelial cells themselves (Millan et al. 1991; Schmid et al. 1991). Both EGF (epidermal growth factor) and TGF ~t are ligands for the EGF receptor (for detection in human lung see Stahlman et al. 1989; John- son et al. 1990), and they have been detected in human fetal and neonatal lung and are believed to initiate terminal cell division with consequent partial induction of the squamous phenotype (Stahlman et al. 1989; John- ston et al. 1990). Growth stimulatory and chemotactic factors that are secreted by the connective tissue may participate in morphogenesis and repair of bronchial epithelium (Jetten et al. 1987) and some factors have been partially purified from human fetal lung and cultured pulmonary fibroblasts (Shoji et al. 1989, 1990b). The recently identified keratinocyte growth factor (KGF) may be another such candidate protein factor (Rubin et al. 1989; Finch et al. 1989). There is some experimental evidence that insulin and insulin-like growth factor-I (somatomedin C) may act as mitogenic chemoattrac- tants on bronchial epithelium (Shoji et al. 1990a). Fur- thermore, insulin-like growth factor-I, shown to be secreted by cultured lung fibroblasts and alveolar macrophages, exerts its effects at an observed physiological concentration (Atkinson et al. 1980; Rom et al. 1988).

The effects of vitamin A have been extensively investi- gated since Wolbach and Howe (1925) reported extensive SCM in the bronchi of rats fed a vitamin A-deficient diet. An overwhelming body of information has accumu- lated on the action and effects of vitamin A on bronchial epithelium (e.g., Wong and Buck 1971 ; Cone and Nettes- heim 1973; Harris et al. 1973; Marchok et al. 1975; An- zano et al. 1980; McDowell et al. 1984a; Jetten et al. 1985, 1987, 1989; Strum et al. 1985; Huang et al. 1986; Jetten and Shirley 1986; Wu and Wu 1986; Stahlman et al. 1988; Rutten etal. 1988a, b, c, d; Jetten 1989; Edmondson et al. 1990; McDowell et al. 1990). The deficiency induced alterations were fully reversible upon replenishment of the vitamin (e.g., Clamon et al. 1974; Lasnitzki and Bollag 1982, 1987; McDowell etal. 1984 b). Vitamin A counteracts the squamous phenotype and instead favors the secretory phenotype (Jetten et al. 1987). Specific nuclear receptors (for expression of human retinoic acid receptor isotypes in lung, see Krust et al. 1989; Leroy et al. 1991 ; Zelent et al. 1991) mediate vitamin A effects on the expression of many genes by interference with their transcription and/or posttrans- criptional processing (for characterization of differentially expressed genes in bronchial epithelium, see Smits et al. 1987). Conversely, vitamin A depletion results in the increased expression of several indicators of stratification and epidermal type differentiation, including certain CKs, transglutaminase type I and sulfotransferase in bronchial epithelial cells (for references see below). In man, vitamin A plasma levels seem to be linked to the frequency of bronchial carcinomas and SCM (e.g., Bjelke 1975; Basu et al. 1976; Mettlin 1979; Wald et al. 1980; Kark et al. 1981; Shekelle etal. 1981; Gouveia et al. 1982; see, however, Willett et al. 1984).

The increased presence of neuroendocrine cells and

neuroendocrine bodies in the bronchial mucosa of the newborn, in patients with chronic pulmonary diseases, and in epithelium next to malignant tumors suggests the involvement of these cells in regenerative processes (e.g., Gould etal. 1983; Addis etal. 1987; Lee etal. 1987). However, we could not find an increased occurrence of such cells using antibodies directed against the broad neuroendocrine marker synaptophysin, an integral membrane protein of the small, clear vesicles of neuroendocrine cells (Wiedenmann and Franke 1985; Lee et al. 1987; Leube et al. 1987). In SCM with marked nuclear atypia and cellular dysplasia, individual neurofi- lament-positive cells have been observed (Moll 1990).

Differences in cell/cell and cell/matrix interactions of polarized and stratified epithelia

The structure and molecular composition of cells in single-layered epithelia differ profoundly from that of suprabasal cells in multilayered epithelia. Thus, cells in one-layered epithelia are structurally and functionally organized in a polar fashion (e.g., Rodriguez-Boulan and Nelson 1989) and this cell polarity is probably mediated and/or kept up by the establishment and maintenance of certain attachment sites which interact either with other cells or with the basal lamina. A crucial step during stratification is therefore the loss of this tpye of polar organization which entails qualitative and quantitative alterations of such attachment structures. These alterations may in turn, not only induce changes of intracellular organization and function, but also act as cues for migration and anchorage. We will therefore describe some of the differences in composition of such attachment sites in different types of epithelium.

One of the recognized hallmarks of epithelial differentiation are desmosomes (maculae adhaerentes) which mediate cell-to-cell contact (for recent reviews see Garrod et al. 1990; Kapprell et al. 1990; Schwarz et al. 1990) and act as anchoring sites for intermediate filament proteins of the CK type in epithelial cells (Franke et al. 1981; Geiger et al. 1983). An increase in number and size of desmosomes has been noted in SCM which were induced either in vitro or in vivo in bronchial epithelium (Gould et al. 1971 ; Wong and Buck 1971 ; Trump et al. 1978; Keenan etal. 1983; Lechner etal. 1984; Jetten et al. 1987), in keeping with the abundance of large desmosomes in the spinous layer of the epidermis. The characterization of several desmosomal polypeptides and their encoding genes shows that some are only present in the desmosomes of stratified epithelia and others are encoded by cell type-specific members of multigene families, thereby demonstrating two principles of adaptation to certain structural and functional requirements im- posed on stratified epithelia. Of the cytoplasmic desmoplakins I and II, which are highly phosphorylated, non- membraneous proteins, and whose encoding mRNAs are possibly transcribed from the same gene by differential splicing (Mueller and Franke 1983; Green etal. 1990; Schwarz et al. 1990), desmoplakin I seems to be an obligatory component of practically all desmosomes

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whereas the somewhat smaller desmoplakin II is only present in some tissues, preferentially in stratified and complex epithelia (Cowin et al. 1985; Green et al. 1990; Schwarz et al. 1990). The significance of alternative splicing of the gene encoding the obligatory plaque protein plakoglobin remains to be shown (Franke et al. 1989). Other cytoplasmic plaque proteins have only been identified in stratified epithelia. These are desmocalmin, a calmodulin- and CK-binding protein detectable in bovine muzzle epidermis and esophagus (Tsukita and Tsukita 1985), desmoyokin, a large protein present in the lateral aspects of the demsosomal plaque in bovine muzzle epidermis (Hieda et al. 1989) and the basic band 6 polypeptide, a facultative protein of maculae adhaerentes in suprabasal cells of certain stratified epithelia (Kapprell et al. 1988, 1990). Band 6 polypeptide has also been detected in several complex epithelia and was detected specifically in the basal cells of rat and bovine tracheobronchial mucosa (Kapprell et al. 1988). Nucleic acid sequence analyses of the constitutive transmembrane glycoproteins desmoglein and desmocollin have identified them as cell adhesion molecules of the cadherin type which are encoded by cell type-specifically expressed members of multigene families (Koch et al. 1990, 1991 a, b; Koch and Franke, personal communication).

The morphologically very similar hemidesmosomes are exclusively present at the basal aspects of epithelial cells (Schwarz et al. 1990; Owaribe et al. 1990, 1991). They interact on the cytoplasmic side with intermediate filaments of the CK type and attach cells to the basal lamina on the other side, presumably by interaction of specific integrins (such as integrin a6f14; Carter et al. 1990; Stepp et al. 1990) with the collagen meshwork through anchoring filaments that are primarily composed of the stratification-related collagen type VII and/ or the recently identified epiligrin complex (Kawanami et al. 1979; Tandler etal. 1981; Ellison and Garrod 1984; Purkis et al. 1990; Carter et al. 1991). The constituent 230kDa bullous pemphigoid antigen and a 180 kDa glycoprotein have been characterized in some detail (Stanley et al. 1988; Klatte et al. 1989; Owaribe et al. 1990, 1991) and antibodies directed against these components specifically decorate the basal aspect of basal cells of stratified and complex epithelia including the bronchial mucosa (Owaribe et al. 1990; Schwarz et al. 1990; Owaribe et al. 1991). It is noteworthy that in contrast to desmosomes, hemidesmosomes are completely absent from simple one-layered epithelia.

Figure 2 gives examples of the differential distribution of desmosomal and hemidesmosomal antigens in fetal trachea, embryonic and adult esophagus and in SCM. In all these instances only the basal aspect of the basal-most epithelial cells are decorated with hemidesmosomal antibodies whereas all cell/cell borders exhibit a spot-like fluorescent staining after reaction with desmosomal antibodies.

Actin filament bundles interact with cell-cell adhesion structures at the apically located belt of zonulae adhaerentes (Geiger et al. 1981) or at similarly composed spot- like puncta adhaerentia which are positioned at the lateral plasma membrane of simple epithelial cells (Drenck-

hahn and Franz 1986). To date, not much is known about compositional variations of these adhesion structures whose constituent polypeptides, plakoglobin, vinculin, c~-actinin, radixin and termin have been characterized (Geiger 1979; Geiger et al. 1981 ; Cowin et al. 1985, 1986; Franke et al. 1989; Tsukita et al. 1989). However, antibodies directed against E-cadherin (= uvomorulin = LCAM =cell-CAM 120/80), an integral transmembrane cell adhesion molecule of the epithelial-type zonulae adhaerentes, show a polarized pattern of immunoreactivity in the human bronchus and trachea, whereas these antibodies decorate the entire cell circumference of suprabasal cells and the apicolateral aspects of basal cells in stratified epithelia such as esophagus and epidermis (the stratum corneum does not react; Eidelman et al. 1989). In addition, its importance in the histogenesis of murine embryonic lung is well documented (Nose et al. 1988; Hirai et al. 1989).

The asymmetric types of actin attachment structures which forms focal contacts in culture have some proteins in common with the zonula adhaerens, such as vinculin and ~-actinin but differ in that they do not contain plakoglobin or E-cadherin and incorporate talin on the cytoplasmic side (e.g., Geiger 1979; Geiger et al. 1981 ; Cow- in et al. 1986; Franke et al. 1989; Eidelman et al. 1989). It is believed that the cell type-specific integrins mediate the interaction of ~-actinin and talin with the extracellular matrix, specifically with fibronectin, laminin and epiligrin (Hynes 1987; Carter et al. 1990, 1991; Ruoslahti 1991).

Other characteristic cell-cell attachment structures determining epithelial cell polarity are the tight junctions of the zonula occludens type that maintain differences in lipid and possibly protein composition in the outer leaflet of the apical and basolateral plasma membranes (Van Meer et al. 1986). The precise tissue distribution of the proteinaceous candidate components ZO-1 (Ste- venson et al. 1986) and cingulin (Citi et al. 1988) are not known but the latter has recently been shown to be a useful marker for adenocarcinomas (Citi et al. 1991). Tight junctions have been detected in bronchial mucosa in a multilaminar necklace arrangement around the base of cilia or microvilli (Elia et al. 1988; Matsu- mura and Setoguti 1989). These junctions probably participate in the maintenance of integrity and polarity of the normal epithelium because a rather weblike and frag- mented arrangement of lamellae has been observed in smokers (Kennedy et al. 1984), and in Syrian hamsters exposed to nitrogen dioxide (Case et al. 1982; Gordon et al. 1986) which may allow access of irritants to reactive cells. Furthermore, in stratified epithelia, and probably also in SCM, tight junctions are completely absent (Elias and Friend 1976).

Electrochemical and metabolic coupling is mediated by specific channel-like structures whose building blocks, the connexins, comprise a multigene family of cell type specific proteins (Dermietzel et al. 1989, 1990; Bennett et al. 1991 ; Meda et al. 1991 ; for expression of connexins 32 and 26 in lung see Zhang and Nicholson 1989). Compositional and positional differences of con- nexons may participate in the subcompartmentalization

234

II . L I L ,, L , L

3--

II,,: ~L;.. "" b~," " ':~:' CT "":" " ~' C .,. : CT

~i ~<~' f" :" L ~ '" ..X '~' L " ., . ,

x. :':.! ' ~ ~...It ~ ~ ~ " r -'", ' - ~ ~"<" "~.u ~ A "

Fig. 2a-|. Photomicrographs of indirect immunofluorescence microscopy using monoclonal hemidesmosomal and desmosomal primary antibodies on various epithelia. (n--c) show fetal tracheal epithelium (gestational week 17) after reaction with antibodies HD- 233, recognizing the hemidesmosomal bullous pemphigoid antigen (a; bracket indicates height of epithelium), DP I/II, specific for desmoplakins I and II (b), and DG 3.10, reacting with demsoglein (e). The same antibodies were applied in parallel on fetal esophagus of the same gestational age as depicted for HD-233 (d; brackets

L

.':~ : : ~ ~A. ie ~, " ~ . ~

3

i CT

on top denote epithelium), DP I/II (e) and DG 3.10 (0. The lower- panel shows HD-233- (g), and DP l/II-reactivity (h) in the adult esophagus in comparison to a bronchial SCM (i; stained with DP I/ II). Note the differential distribution of the much smaller hemidesmosomes at the interphase between basal cells and basal lamina in these tissues and the abundant desmosomes, decorarating lateral and apical surfaces of basal cells and the entire circumference of suprabasal cells. C T , connective tissue; L, lumen. Bars, 50 p.m

235

. . t ~ ,

'

& . - .

~: C T ~ ,-

Fig. 3a--g. Indirect immunofluorescence microscopy of human bronchus and esophagus reacted with monoclonal antibodies against CKs 5 and 17. a--d depict staining of CK 5-reactive antibody AE14 on 5 pm tissue sections from bronchial mucosa (a; the left third shows region of hyperplasia), SCM (b) and esophagus of adult (e)and fetus (d; gestational week 17). Brackets denote in (a) the entire epithelial thickness on the right, the basal cell layer on the left margin and in (tl) the entire epithelium. (e-g) show the reactivity of CK 17-specific antibody E3 with human bronchus (e; top half contains nomal epithelium in inverted posi-

tion opposite to SCM in the buttom part in upright position), adult esophagus (f) and fetal esophagus (g; gestational week 17). The broken lines in (e) and (f) indicate position of basal lamina as determined by phase contrast optics. The brackets in (e, g) denote epithelium. Note the labeling of basal cells with AE14 in all instances whereas E3 stains only very few cells in SCM and adult esophagus in contrast to the basal cell reactivity of normal bronchial epithelium and developing esophagus. L, lumen; CT, connective tissue. Bars, 50 p.m

236

of stratified epithelia (Elias and Friend 1976; Kam et al. 1986). Furthermore, it has been described that gap junctions are only rarely detected in the basal cell compartment of the normal rat tracheas, but are abundant in conditions of high proliferative activity (Gordon et al. 1982).

It is interesting in the context of epithelial plasticity, as encountered in SCM, that junctions can be rapidly endocytosed in toto upon removal of extracellular calcium, and that replenishment of calcium facilitates the re-integration into the plasma membrane from presump- tive intracellular vesicles (e.g., Kartenbeck et al. 1982, 1991; Mattey and Garrod 1986; Duden and Franke 1988). Similar mechanisms may be employed in the de- tachment of an epithelial cell from the basal lamina and neighboring basal cells as it migrates upward (Watt et al. 1984). The calcium-dependency of epithelium/substratum adherence has been exploited for the efficient separation of bronchial epithelial cells from the basal lamina using 20 mM EDTA which reportedly induces internalization of hemidesmosomes (Tandler et al. 1981).

The cell adhesion molecules of the cadherin type (cell/ cell recognition) and integrin type (cell/extracellular matrix attachment) occurring either as constituents of the above mentioned electron microscopically defined structures or as free floating plasma membrane components regulate migration, adhesion and recognition and couple these processes to the cytoskeleton (for recent reviews see Ruoslahti 1991; Takeichi 1991). These molecules interact with the intracellular plasmalemmal lattice which is composed of proteins such as fodrin and ankyrin (Nel- son and Veshnok 1987a, b; Nelson et al. 1990). There- fore, the distribution and assembly state of the intracellular framework has been shown to be dependent on the relative localization within stratified epithelia such as epidermis (Yoneda et al. 1990). In accordance, the type of substratum has been demonstrated to influence the differentiation of bronchial epithelial cells in culture (Jetten et al. 1987).

Distribution of cytokeratin polypeptides in normal human bronchial mucosa

Certain cues from cell/cell and cell/extracellular matrix interactions are transmitted to the cytoskeleton which is the organizing framework within the cell that coordi- nates compartmentalization, cell shape and mobility. Of the three major filament systems that of intermediate size is probably the most complex in terms of protein composition. Two large multigene families encoding the smaller, more acidic type I and the larger, more basic type II CK polypeptides are expressed in specific combi- nations in epithelial tissues (for review see Moll et al. 1982a; Cooper et al. 1985; Quinlan et al. 1985; Franke 1987; Fuchs 1988). Therefore, reagents detecting individual CKs are extremely useful tools in the characterization of epithelial morphogenesis and differentiation.

The archetypic CKs 8 and 18 are obligatory components of the single-layered epithelia of stomach, intes-

tine, liver, pancreatic acini or pulmonary alveoli where all cells abut the basal lamina and lumen (e.g., Ra- maekers et al. 1983; Blobel et al. 1984; Leube et al. 1986; Woodcock-Mitchell et al. 1989). In addition, some of these epithelia may also express CKs 7, 19 and 20 (Ramekers et al. 1983; Woodcock-Mitchell et al. 1989; Moll et al. 1990). In contrast, the cellular heterogeneity of complex epithelia of the urogenital tract and of glandular structures with various specialization is reflected by a wealth of differentially expressed CKs (e.g., Czerno- bilsky etal. 1984; Achtst/itter et al. 1985; Levy et al. 1988; Nagle et al. 1986; Wernert et al. 1987; Gueistein et al. 1988; Schaafsma et al. 1989; Troyanovsky et al. 1989; Purkis et al. 1990; Sherwood et al. 1990). The pattern of CK expression in these tissues supports the notion that they share certain differentiation features not only with simple epithelia but also with stratified epithelia. Thus, in most instances adluminal, columnar cells show the "simple" phenotype (CKs 7, 8, 18 and 19) whereas basal cells express CKs 5 and 14 that are also characteristic of basal cells in multilayered epithelia (e.g., Nelson and Sun 1983; Bosch etal. 1988; Lersch and Fuchs 1988; Purkis et al. 1990). The bronchial mucosa is a complex epithelium requiring a more detailed de- scription of regional expression of CK polypeptides.

The human tracheobronchial epithelium contains the simple epithelial-type CKs 7, 8, 18, 19 and minor amounts of CK 20 together with CKs 5, 6, 14, 15 and 17 (Bejui-Thivolet et al. 1982; Moll et al. 1982a, 1989; Blobel et al. 1984; Wilson et al. 1985; Banks-Schlegel et al. 1984; Lehto et al. 1986; Van Muijen et al. 1986; Broers et al. 1988, 1989; Troyanovsky et al. 1989; Moll 1990; for other species, cultured cells and human xeno- transplants see Keenan et al. 1983; Gilfix and Eckert 1985; Jetten and Smits 1985; Huang et al. 1986; Klein- Szanto et al. 1986; Wu and Wu 1986; Obara et al. 1988; Rutten et al. 1988c, d; Huang et al. 1989; Jetten et al. 1989). Using specific antibodies a differential distribution of these polypeptides is discernible, thereby allowing the gross distinction of a basal and luminal phenotype.

Thus, in human, CKs 8 and 18 together with CKs 7 and 19 can be identified in all adluminal cells indepen- dent of their particular specialization (Blobel et al. 1984; Lehto et al. 1986; Broers et al. 1988, 1989). However, these CKs are not absolutely restricted to this cell type and may also be detected - albeit at a lower level - in basal cells, depending on the tissue section, patient or antibody. Similarly, their expression is not completely shut off in stratified epithelia of the esophagus, exocervix or vagina (e.g., Franke et al. 1986, 1987b; Bosch et al. 1988). Antibodies against the recently identified CK 20 only stain a few individual adluminal cells in human lung (Moll 1990).

The basal-type CKs 5 and 14 of stratified and complex epithelia are also present in the basal cell layer of the bronchial epithelium (Fig. 3 a; see also Blobel et al. 1984; Wilson et al. 1985; Lehto et al. 1986; Broers et al. 1988, 1989; Moll et al. 1989; Purkis etal. 1990). It is of interest that these basal cells as well as their counter- parts in stratified epithelia are both attached to the basal lamina by hemidesmosomes which may specifically in-

237

Fig. 4a-f. Epifluorescence photomicrographs of bronchial mucosa (a--e) and esophagus (f) reacted with CK antibodies (a, b) show double fluorescence of bronchial mucosa stained with affinity-purified CK 15 antibodies raised in the guinea pig (gp 15.1; a) and the broad-reactive CK antibody lu-5 (b; the detail is taken from a study by Heid, Bartek, Leube, Moll, Kaufmann and Franke, in preparation). The same basally restricted fluorescence is seen

: L_

C CT

in regions with mucous cell hyperplasia after reaction with gp 15.1 as primary antibody (e; bracket indicates entire epithelial thickness). In SCM, however, all epithelial cells are stained with gp 15.1 as demonstrated in d (e, corresponding phase micrograph to d). The reaction of all epithelial cells with these antibodies is also found in the esophagus (f). CT, connective tissue; L, lumen. Bars, 50 pm

teract with CKs 5 and 14. The detection of CKs 15 and 17 is similarly restricted to basal cells of bronchial epithelium (Figs. 3e and 4a, c; see also Troyanovsky et al. 1989). These CKs show, however, a different tissue distribution from each other and from CKs 5 and 14 in other epithelia: i) CK 17 is only present in individual basal cells of esophagus (Fig. 3f; for increased expression in basal cells of fetal esophagus see Fig. 3g) and absent from epidermis except when its expression is induced under certain proliferative conditions such as psoriasis vulgaris or fetal development together with CKs 6 and 16 (Leigh et al. 1985; Stoler et al. 1988; De Jong et al. 1991; Troyanovsky, personal communication); ii) in contrast CK 15 is detectable in all epithelial cell layers of "non-keratinizing" epithelia such as the esophagus (Fig. 4f; see also Leube et al. 1988) and only in a topi- cally restricted pattern in epidermis (Heid and Leube, unpublished observations).

The hallmark proteins of stratified, non-keratinizing epithelia are CKs 4 and 13 where their expression is restricted to suprabasal cell layers (e.g., Cooper et al.

1985; Franke et al. 1986; Van Muijen et al. 1986; Leube etal. 1988; Kuruc et al. 1989; Bosch etal. 1989). In bronchial mucosa, antibodies to CKs 4 and 13 react only with individual cells or cell clusters (Van Muijen et al. 1986; Broers et al. 1988, 1989). One interpretation is that these heterogeneities arise from environmentally induced alterations rather than constitutive lack in precise gene regulation in bronchial epithelial cells. Follow- ing this rationale the detection of these polypeptides would be an extremely sensitive indicator of epithelial change that can not be identified by conventional histomorphological techniques.

Keratinizing epithelia such as epidermis are characterized by CKs 1/2 and 10/11 that are presumably induced upon committment to terminal differentiation (e.g., Fuchs and Green 1980; Moll et al. 1982b; Stoler etal. 1988). Accordingly, CKs 1 and 10/11 were not detected in normal-appearing bronchial mucosa (Broers etal. 1988; Rustad, Leube, Moll, Bosch and Fran- ke, in preparation; see, however, B6jui-Thivolet et al. 1982).

238

Characteristic changes of cytokeratin expression in squamous cell metaplasia of the human lung

SCM of the lung is a well suited model for investigating the changing CK expression in a pathologically relevant situation. The validity of this type of approach has been demonstrated by examining CK expression during stratification accompanying development (e.g., Moll et al. 1982b; Lane et al. 1985; Van Muijen et al. 1987) or SCM formation of the uterine cervix (Puts et al. 1985; Gigi- Leitner et al. 1986; Huszar et al. 1986; Molloy and Las- kin 1988; Levy et al. 1988) and the urothelium (Tungek- ar et al. 1987, 1988; Fukushima et al. 1987; Moll et al. 1988; Schaafsma et al. 1989, 1990). The first systematic investigation of CK expression in human bronchial SCM (Rustad, Leube, Moll, Bosch and Franke, in preparation) extending observations in certain model systems (Keenan et al. 1983; Gilflx and Eckert 1985; Jetten and Smits 1985; Wilson et al. 1985; Huang et al. 1986; Klein- Szanto et al. 1986; Wu and Wu 1986; Obara et al. 1988; Rutten et al. 1988c, d; Huang et al. 1989; Jetten et al. 1989) will be summarized briefly.

The expression of the simple-type CKs which are most abundant in the columnar adluminal cells of normal bronchial epithelium were greatly reduced in all SCM, albeit to a rather variable degree (Fig. 5 a). Thus, CKs 7 and 20 were not detected in any SCM (see also Moll 1990) whereas CK 19 expression was obviously retained in many cells, sometimes in all metaplastic cell layers. CKs 8 and 18 antibodies did not react with cells within a given metaplastic focus but stained individual cells either at the surface or in certain intraepithelial "islands" (Fig. 5). Interestingly, SCM with marked nuclear atypia and dysplasia reacted rather strongly with these antibodies (see also Moll 1990).

The expression of the basally located CKs was altered in different ways during metaplasia formation. CKs 5 and 14 were present at almost the same relative amounts and could be detected only in the basal-most cell layer(s) of SCM (Fig. 3a, b). The expression of CKs 6, 15 and 16 was greatly induced as judged from the intense immunoreactivity of all cell layers with specific antibodies, increased polypeptide detection in gel-electrophoretic analyses of cytoskeletal proteins from microdissected metaplastic bronchial epithelial cells, and increased mRNA levels (Fig. 4d, e; see also Obara et al. 1988). In marked contrast, only a few individual cells were stained with CK 17 antibodies in metaplastic foci (Fig. 3e). Despite the divergence of expression of all these basal-type CKs, their distribution in SCM rather closely reflects changes expected upon transformation to a stratified epithelium as shown by the comparative fluorescence micrographs of human esophagus in Figs. 3 c, f and 4 f.

Stratification-related CKs 4 and 13 were abundantly expressed in SCM in contrast to their limited detection in certain areas of normal-appearing epithelium (Fig. 6 a, b). Again, the characteristic suprabasal distribution is indistinguishable from that observed in the esophagus (Fig. 6 c, d).

In conclusion, the CK composition of all cells within

SCM differs from that of cells in normal bronchial epithelium. This demonstrates that a true change in tissue differentiation has taken place which, more importantly, indicates that a preceding alteration of the differentiation character of the potential stem or progenitor cells must occur.

The uniformity of CK expression changes in SCM can be taken as an indication of the limited complexity of developmental options which might therefore be controlled by a small number of master genes. Master genes encode transcription factors that coordinate specific pro- grams such as myogenesis (for recent review see Wein- traub et al. 1991), and epithelial differentiation can be presumed to have analogous key regulators. To date, very little is known about such regulators in stratified epithelia (Leask et al. 1990; Snape et al. 1990). The clus- tered organization of CK genes seems to point to some general principle of coordinate CK gene transcription (for man see Blumenberg and Savtchenko 1986; Ray- Chaudhury et al. 1986; Bader et al. 1988; Rosenberg et al. 1988; Popescu et al. 1989; Savtchenko et al. 1990; Troyanovsky, Leube and Franke, unpublished data). Yet, individual cis-acting elements that confer cell type- specific expression have been identified for a number of human CK genes (e.g., Lersch et al. 1989; Vassar et al. 1989; Bader et al. 1990; Snape et al. 1990). Furthermore, CK gene expression is also affected by methylation (e.g., S6mat et al. 1986; Knapp and Franke 1989) and the mRNA levels of several CKs are influenced by vitamin A which inhibits SCM (e.g., Kim et al. 1984; Eckert and Green 1984; Gilfix and Eckert 1985; Kim etal. 1987; Kopan and Fuchs 1989).

The relationship between CK expression and proliferation is still rather obscure. CKs 8 and 18 are expressed in epithelia with high proliferative activity and are present in most, if not all carcinomas (e.g., Bosch et al. 1988; Cheng et al. 1990; Moll 1990). Their expression may even be induced in certain immature mesenchymal tissues during embryogenesis or by hypomethylating agents (e.g., Jahn et al. 1988; Kuruc et al. 1988; Knapp and Franke 1989). Our data provide no evidence for coordination of growth and CK expression in human bronchial epithelium since co-segregation of specific CK reactivity and Ki-67 positivity was not observed (for contrasting interpretation see however, Obara et al. 1988).

Development of the stratified epithelium of esophagus as a paradigm for squamous cell metaplasia

The epithelium of the tracheobronchial tract and the esophagus both originate from the endoderm (for details see Enterline and Thompson 1984; Boyden 1972). This fetal epithelium consists of a single polarized layer of cuboidal cells. Gradually, a second cell layer develops and a two-layered columnar pseudostratified epithelium is detectable by the 6th week of gestation. Around the 10th week goblet and ciliated columnar cells can be seen in the adluminal compartment. At this time, further development seems to arrest in the mesenchyme-invested bronchial buds, whereas the prospective esophagus de-

239

Fig. 5a-c. Immunofluorescence micrographs of cryostat sections from SCM (a) and fetal esophagus (b, c) after reaction with monoclonal antibodies directed against the simple-type CKs 8 and 18. The composite photomicrograph in (a) shows metaplastic bronchial epithelium (horizontal brackets) next to non-metaplastic mucosa stained with the CK 8-specific antibody Ks8.17.2. The broken line indicates the position of the basal lamina. (b, e) Pattern of reaction

of the CK 8-specific antibody Ks8.17.2 (b) and the CK 18-specific antibody Ks18.174.1 (c) with fetal esophagus (gestational week 17). Note the strong fluorescent labeling of individual adluminal cells in (a-c). Weak positivity of other epithelial cells in fetal esophagus as seen in (b) and e may persist to adulthood wheras columnar adluminal cells will not be retained (Bosch et al. 1988). L, lumen; CT, connective tissue. Bars, 50 p.m

velops first into a transitional-type epithelium and finally into the mature stratified squamous surface layer. At birth, only a few remnant islands of ciliated pseudostratified epithelium are present in the esophagus. These observations originally led to the interpretation that the development of the bronchial epithelium is arrested at a specific point by unknown mechanisms and that SCM represents a derepression of these restrictive mechanisms (Schridde 1907, 1909; Teutschlaender 1919).

With specific biochemical probes we can now reexam- ine this question by comparing the distribution of certain markers during esophageal development and generation of SCM. Our own results strongly support the notion that the bronchial mucosa may reenter an intrinsic pathway of differentiation that is normally repressed (Figs. 2 6). Striking similarities are observed in CK expression between fully developed SCM and esophageal epithelium: the abundance of CKs 4 and 13 in supraba-

240

sal cell layers (Fig. 6), the detection of CKs 5 and 14 in basal cells (Fig. 3), the rare staining of basal cells with CK 17 antibodies (Fig. 3), and the detection of CK 15 in all epithelial cell layers (Fig. 4). Furthermore, both tissues exhibit the same type of differential distribution of hemidesmosomes and desmosomes as depicted in Fig. 2. The immunofluorescence micrographs of Fig. 5 suggest that similar dynamic processes take place during development of esophagus epithelium and SCM as columnar CK 8- and 18-positive cells are detected in both instances at the adluminal surface, as if they were pushed upwards by a basal to luminal proliferative momentum (the significance of the somewhat variable and weak reactivity of cells in lower strata of the fetal esophagus in Fig. 5 remains to be shown).

Similarities of epidermal differentiation and squamous cell metaplasia of the human lung

As SCM may develop even further into lesions which phenotypically resemble epidermis, accompanying changes in gene expression should be expected in these foci.

Thus, we observed the ectopically induced expression of CKs 1 and 10/11 in SCM (see also B6jui-Thivolet et al. 1982). These CKs were detectable prior to the onset of overt cornification as they gradually substitute for the suprabasal esophageal-type CKs 4 and 13 (a thor- ough presentation of the detection of these CKs and their encoding mRNAs will be given in the paper by Rustad, Leube, Moll, Bosch and Franke).

The abundance of desmosomes in SCM as the hallmark feature of the stratum spinosum has been mentioned before and is shown in Fig. 2i.

One of the polypeptides, specific for the "granular" layer, is the histidine-rich protein filaggrin that is probably a major component of the electron microscopically dense-appearing keratohyalin granules (Dale 1977; Dale etal. 1978; Lynley and Dale 1983; Fleckman etal. 1985). Keratohyalin-like granules have been noted in SCM (Wong and Buck 1971; Marchok etal. 1977; Klein-Szanto et al. 1980c; Chopra 1982) but the presence of filaggrin in SCM has not yet been confirmed with specific antibodies. Cholesterol sulfate is highly en- riched in the stratum granulosum of skin and it may accumulate up to 100 times in bronchial epithelial cells cultured in vitamin A-depleted media (Rearick and Jet- ten 1986; Rearick et al. 1987).

In the stratum corneum, cells filled with insoluble CK filaments attached to basic proteins such as fillagrin are first engaged in the formation of the cross-linked envelope, then lose their nucleus and die. A few constitutent polypeptides of the mechanically very resistant intracellular subplasmalemmal envelope structure have been well described: i) involucrin, a protein composed of short repetitive units that are rich in lysine and glutamic acid which are extensively linked with the help of the specific transglutaminase type I (Rice and Green 1979; Banks-Schlegel and Green 1981; Eckert and Green 1986); ii) loricrin, a sulfur-rich protein that is stabilized

by extensive disulfide bonds (Mehrel et al. 1990); and iii) a basic 25 kDa protein (O'Guin et al. 1989). Involuc- rin and possibly other envelope proteins can be expressed by bronchial squamous metaplastic cells and certain pulmonary carcinoma cells (Said etal. 1983b; Banks-Schlegel etal. 1985; Klein-Szanto etal. 1986; Lehto et al. 1986; Ke et al. 1990). Furthermore, a 20- to 30-fold increase in type I transglutaminase activity has been detected in terminally differentiated cultured rabbit tracheal epithelial cells (Jetten and Smits 1985; Jetten and Shirley 1986; Jetten et al. 1987). This enzyme is absent in normal rat tracheobronchial epithelium (Par- enteau et al. 1986).

Calcium ions may play a crucial part in terminal differentiation as they enhance transglutaminase and cholesterol sulfate expression, and the formation of cross- linked envelopes in cultured bronchial epithelial cells (Jetten and Shirley 1986; Rearick and Jetten 1986; Rear- ick et al. 1987).

Histogenesis of squamous cell metaplasia

To date, evidence has accrued for practically all alternative modes of metaplasia formation in the lung. It may therefore be true that alternative pathways may involve different cell types, each of which can transdifferentiate into squamous cells. If this is true, a histogenetic classification is far inferior to a classification based on specific differentiation markers.

Virchows' proposed concept of direct metaplasia has survived in a somewhat modified form in the model put forward by Trump, McDowell and coworkers (Becci etal. 1978a, b; Trump etal. 1978; McDowell etal. 1978b) who postulated a direct conversion of mucous- secreting cells into squamous cells as they find small mucous granules in cells of SCM. However, it is quite possible that the nature of these vesicles detected in SCM is quite different from those in small mucous granule cells (e.g., for occurrence of "small vacuoles" see Wong and Buck 1971 ; Wang et al. 1972).

More recently the concept has been somewhat modified such that it was proposed that small mucous granule cells, a type of undifferentiated immature cells, first proliferate (a process termed mucous cell hyperplasia or the phase of neoplasia) and then transform into squamous cells (the phase of metaplasia; e.g., McDowell et al. 1980, 1984). The observation that some cells in SCM show features intermediate between columnar and squamous cells was taken as evidence for this model (McDowell et al. 1980, 1984a; Sigler et al. 1988). This hypothesis, however, does not rule out the participation of basal cells in SCM, which cannot be excluded because incorporation of tritiated thymidine and mitoses were also observed in basal cells (e.g., Keenan et al. 1982a, b; McDowell et al. 1984a, b; Strum et al. 1985).

A number of differentiation markers were used by Niimi et al. (1987) to elucidate the histogenesis of SCM in man. They interpret the detection of "glandular" markers, secretory component, epithelial membrane antigen, and carcinoembryonic antigen in SCM as an indi-

241

Fig. 6a-d. Comparison of the pattern of fluorescent staining of SCM (a, b) and esophagus (e, d) using primary antibodies directed against the stratification-related CKs 4 and 13. (a, b) Photomicro- graphs of SCM after reaction with CK4-specific antibody 6B10 (a) and CK13-reactive antibody Ks13.1 (b). The fluorescence mi-

crographs in (e, d) show human esophagus after reaction with the same antibodies (6B10 in e, Ksl3.1 in fl). Note the strong suprabasal labeling in all tissue samples. The relative position of the basal lamina is denoted by a broken line. CT, connective tissue; L, lumen. Bars, 50 ~tm

242

cation of mucousal cell derivation. Yet, their own observations that the 7-type enolase which is usually present in luminal cells of normal epithelium was absent from SCM, and that the c~-type enolase which is predominantly detectable in basal bronchial epithelial cells showed an increased expression in SCM, conflict with their interpretation.

Probably the most popular model discussed today is the participation of pluripotent basal cells (the stem cell model). Originally, descriptive observations led to the notion that basal cells proliferate and thereby push luminal, columnar cells upwards (Teutschlaender 1919; Wong and Buck 1971; StenNick 1973; Chopra 1982; Chopra and Cooney 1985; Harris et al. 1989). These latter cells or the descendants of cells which replace the shedded cells, slowly lose their peculiar surface features as they gradually attenuate and accumulate large amounts of tonofilaments (Reznik-Schiiller 1980; Yama- moto et al. 1987). It was shown experimentally that basal cells can act as stem cells since they are able to differentiate into ciliated cells, goblet cells, and secretory cells with small granules (Inayama et al. 1988) but that these cells differentiate into squamous cells in vitamin A deficiency thereby giving rise to SCM (Lane and Gordon 1979). Furthermore, somewhat in contrast to the above mentioned studies, Chopra (1982) reported the predominant labeling of basal cells with tritiated thymidine in cultures of hamster tracheas under vitamin A deprivation, most notably at the protruding edges of SCM. Most studies addressing the problem of the histogenesis of SCM using various markers agree that none of the cells in a given metaplastic focus is completely identical to cells of the normal epithelium. Apically located cells often display features of adluminal cells such as the expression of secretory component, epithelial membrane antigen, carcinoembryonic antigen or CKs 8 and 18, but such cells are completely absent from highly stratified SCM (Fig. 5; Niimi etal. 1987; Yamamoto etal. 1987). We showed that basal cells in SCM differ from those of normal epithelium by lack or reduction of expression of CK 17 (Fig. 3e). Furthermore, suprabasal cells differ from adluminal cells of the normal bronchial epithelium by the neoexpression of CKs 4, 13 and 15 or in some instances even of CKs 1 and 10/11 (Figs. 4 and 6; Moll 1990). These observations suggest that certain progenitor cells not only give rise to altered progeny but are themselves altered in that they exhibit certain new differentiation features which may indicate a crucial step in transdifferentiation.

The stem cell model does not contradict the notion that SCM is a continuation of an intrinsic program of normal development whose endpoint could be epidermal differentiation (Schridde 1907, 1909; Wittekind and Strfider 1953b; Jetten 1989). However, the high labeling index of "mature" metaplastic cells speaks against this simple view (Fig. 1 ; Chopra 1982).

The reversibility of SCM in the human lung has long been propsoed (e.g., Wittekind and Str/ider 1953b) and been shown convincingly in several model systems to occur after removal of the irritant either spontaneously or upon vitamin A supplementation (Clamon et al.

1974; Becci et al. 1978a; McDowell et al. 1980; Moss- man et al. 1980; Keenan et al. 1982a, b, c; Lasnitzki and Bollag 1982; Keenan et al. 1983; McDowell et al. 1984b; Rutten et al. 1988c; Cameron et al. 1989). A limited number of clinical studies demonstrate the protec- tive effects of vitamin A or analogues, folate and vitamin B12 on the formation of tumors and SCM in smokers (Misset et al. 1986; Heimburger et al. 1988).

In conclusion, a classification of pulmonary alterations - be they of benign or malignant nature - should not be based on possible, and often questionable histogenetic relationships but rather on specific differentiation features. The detection of a number of these markers should be suitable to show the dynamic nature of the stratification process with frequently overlapping phenotypes, and should be a very precise indication of biological behavior to determine therapeutic action.

Squamous cell metaplasia and tumor formation in human lung

One of the earliest reports on the relationship of SCM and squamous cell carcinoma (SCC) described the grad- ual transition of the tumor tissue into the thickened, "pachydermic" bronchial epithelium in four patients with SCC of the lung (Watsuji 1904). Despite the fact that SCM is more common in patients with pulmonary neoplasms than in control groups, a heated and still unresolved discussion has continued over the years about the possible causal relationship between SCM and malignant transformation (e.g., Lindberg 1935; Black and Ackerman 1952; Wittekind and Strfider 1953b; Weller 1953; Valentine 1957; Nasiell 1966; McDowell et al. 1978b; Trump et al. 1978; Tsuchiya et al. 1987). Therefore, some authors attempted to subdivide SCMs according to varying degrees of atypia and morphology (Auerbach et al. 1961, 1962; for atypia in exfoliated cells see Saccomanno et al. 1974; Nasiell 1966; Johnston 1986; Vine et al. 1990), each correlating to particular steps in the multistage process of carcinogenesis (Pfeifer et al. 1989). Animal studies indicate that the type of chronic environmental irritation and/or the specific combination with other inducers of SCM is crucial for the development of malignancies (e.g., Saccomanno et al. 1970; Klein-Szanto et al. 1981; Woodworth et al. 1983; Keenan et al. 1989). The identification of dark cells in experimentally induced metaplasias describes another at- tempt to differentiate certain types of SCM (Klein-Szan- to et al. 1980a, b, c, 1981 ; Nettesheim et al. 1982). Other authors propose that dysregulation of terminal differentiation pathways may be closely linked to neoplastic transformation in agreement with the observed resis- tance of lung carcinoma cells to inducers of terminal differentiation (Willey et al. 1984; Masui etal. 1986; Pfeifer et al. 1989) and the differences in responsiveness of bronchial epithelial cells transfected with the SV40 large T antigen to TPA, TGF fll and fetal bovine serum (Ke et al. 1988). We noted that increasing nuclear atypia and cellular dysplasia are paralleled by a loss of ordered CK expression and rather bizarre patterns of CK co-

243

r

i

:4

Fig. 7a--g. Differential reactivity of specific antibodies directed against desmosomal antigens and CKs in a selected squamous cell carcinoma of the lung as seen by epifluorescence microscopy. Stain- ing is shown for antibodies DP I/II against desmoplakins I and II (a), Ks8.17.2, specific for CK8 (b), Ks18.174.1, reacting with

CK 18 (c), 1C7, directed against CK 13 (d; e corresponding phase micrograph) and Kk8.60 against CKs 10/11 (f; g corresponding phase micrograph). Note, that except for DP I/II none of the antibodies reacts with all tumor cells indicating heterogeneity of tumor differentiation. Bars, 50 I.tm

expression were encountered, possibly indicating uncou- pled control of specific epithelial differentiation pro- grams (Moll 1990; Rustad, Leube, Moll, Bosch and Franke, in preparation).

Many authors accept the possible involvement of SCM in the generation of squamous cell carcinomas of the human lung (e.g., Watsuji 1904; Lindberg 1935; Black and Ackerman 1952; Valentine 1957; Nasiell 1966; Trump et al. 1978; Tsuchiya et al. 1987) but the situation for other types of lung tumors is much less clear.

Several marker proteins that are detected in SCM are also present in SCC and can aid in the differential diagnosis of pulmonary neoplasmas (Fig. 7; Said et al. 1983a, b; Banks-Schlegel et al. 1984; Blobel et al. 1984; Wilson et al. 1985; Lehto et al. 1986; Obara et al. 1988). As the SCC character of pulmonary neoplasms is associated with slow growth rates and therefore a better prognosis and a different therapeutic regimen, these indicators of stratification may be very useful adjuncts in the evaluation of lung tumors. Since stratification

244

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~. , . ~ 2 . . ' l i t ~ '.t ! ~ i , i : " 4, ~ ,o , , , ~ . !

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�9 �9 , . . ~ . , . - ;.,,. ~, . .~ ~ . ~ . - f f ~ . - x ~ : .~ - . ' . ,

�9 . : ~ - , - - . ' - ~ ' ~ ~ ; 9 ~ , ~ : ~ , ~ t : ~ , ' , ~ : "

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... ~ . . ~ ~ . . * '~ %.. , : , ~ "~ ~ . : ~ , ~ . , ' ~ , ~ . . ~ ~ ~"

Fig. $,,-j. Detection of intermediate filament polypeptides by immunofluorescence microscopy in cells obtained from bronchial washings. Desquamated bronchial cells were spread on glass slides and fixed with methanol/acetone�9 (~-e) show double labeling of cells with monoclonal CK 4-antibody 6BI0 (h) and broad-reactive CK antiserum gp 10 from guinea pig (e; a corresponding phase micrograph). (d, �9 depicts the positive staining of alveolar macrophages with monoclonal anti-vimentin antibody Vim-3B4 (d fluo-

K

k.

rescence microscopy; (e) corresponding phase contrast). Note the absence of vimentin-reactivity in ciliated cells, if-k) show rarely occurring cells reacting with either polyclonal CK 15 antibodies gplS.1 (g; f phase contrast) or monoclonal CK 13 antibody Ksl3.l (i; h phase contrast) and CK 4 antibody 6B10 (k ; j phase contrast). (I) shows the reactivity of CK 8 antibody M20 with all epithelial cells seen in this field. Bars, 25 ~tm

markers may be present in different proportion of various pulmonary neoplasms (e.g., Fig. 7; Said et al. 1983a; I.ehto el al. 1986; Broers et al. 1988), quantitative measurements of these parameters may be en- visioned which will allow a biochemical "grading". Fig- ure 8 demonstrates the usefulness of CK antibodies alone or in combination for the staining of routine cy- tospin preparations obtained from bronchial lavage. Therefore, similar cytological specimens should be suitable for analyses in automated assays, and may ultimate-

ly replace painstaking and very subjective evaluation procedures (e.g., NasieU 1963; Saccomanno et al. 1965, 1974; Nasiell 1966; Schreiber et al. 1974; Johnston 1986; Vine et al. 1990).

Acknowledgements�9 We greatfully acknowledge the expert technical assistance of C. Kuhn and S. Stumpp. We are also indepted to the generous provision of human tissues by Drs. K. Kayser and H.-G. Manke and thank Dr. W.W�9 Franke for continous support and Drs. M. Murray and S. Troyanovsky for critical reading of the manuscript.

245

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