Cell type heterogeneity of cytokeratin expression in complex epithelia and carcinomas as demonstrated by monoclonal antibodies specific for cytokeratins nos. 4 and 13

Three monoclonal antibodies, 1C7, 2D7 and 6B10, directed against cytokeratins of human esophagus were isolated and characterized by one- and two-dimensional gel electrophoresis and by immunohistochemical staining on sections of human epithelial tissues. In immunoblot experiments, antibodies of clones 1C7 (IgG2a) and 2D7 (IgG2b) react only with cytokeratin no. 13 of the acidic (type I) subfamily of cytokeratin polypeptides (Mr 54000; pI 5.1); antibodies of clone 6B10 (IgG1) detect only cytokeratin no. 4 (Mr 59000; pI 7.3) of the basic (type II) cytokeratin subfamily and allows the detection of this protein and possible degradation products at high sensitivity. In immunohistochemical staining all three antibodies stain non-cornifying squamous epithelium (e.g., tongue, esophagus, anus) and transitional epithelium of the bladder. Antibodies of clone 6B10 also stain cells in certain ciliated pseudostratified epithelia and ductal epithelia of various exocrine glands. These monoclonal antibodies are the first examples of antibodies specific for individual cytokeratin polypeptides characteristic of certain complex epithelia. They allow the identification of distinct minor populations of cells present in certain complex and glandular epithelia and in tumors derived therefrom which hitherto have not been distinguished. The possible reasons for the occurrence of cell type heterogeneity of cytokeratin expression in complex epithelia and in some carcinomas are discussed.     

Changes in the distribution of intermediate filament proteins and collagen IV in fetal and adult human pancreas. I. Localization of cytokeratin polypeptides

The expression patterns of individual cytokeratin polypeptides in foetal and adult human pancreatic tissues were examined using monoclonal antibodies. We demonstrated that human pancreatic epithelia in early stages of development (14 weeks of gestation) contain cytokeratins 7, 8, 18 and 19, which are typical of simple epithelia, as well as cytokeratin 4 and 17, which are characteristic of stratified epithelia. In the pancreatic ducts, most of these cytokeratins appeared to be expressed together. Cytokeratins 1, 5, 10, 13, 16 and 20 were not detectable. In contrast, the pancreatic parenchyma was only positive for cytokeratins 8 and 18, except a transient expression of cytokeratins 7 and 19 in pancreatic islets and acinar cells during the foetal development. A focal cytokeratin 7 staining of single acinar cells was seen in newborn and in adult islets. In the stromal tissue, vascular smooth muscle cells were partly reactive with cytokeratin 8 and 18 specific antibodies. The results are discussed in the light of differentiation-dependent changes in the expression of individual cytokeratin polypeptides in developing epithelia.     

Changes in the distribution of intermediate filament proteins and collagen IV in fetal and adult human pancreas

Summary The expression patterns of individual cytokeratin polypeptides in foetal and adult human pancreatic tissues were examined using monoclonal antibodies. We demonstrated that human pancreatic epithelia in early stages of development (14 weeks of gestation) contain cytokeratins 7, 8, 18 and 19, which are typical of simple epithelia, as well as cytokeratin 4 and 17, which are characteristic of stratified epithelia. In the pancreatic ducts, most of these cytokeratins appeared to be expressed together. Cytokeratins 1, 5, 10, 13, 16 and 20 were not detectable. In contrast, the pancreatic parenchyma was only positive for cytokeratins 8 and 18, except a transient expression of cytokeratins 7 and 19 in pancreatic islets and acinar cells during the foetal development. A focal cytokeratin 7 staining of single acinar cells was seen in newborn and in adult islets. In the stromal tissue, vascular smooth muscle cells were partly reactive with cytokeratin 8 and 18 specific antibodies. The results are discussed in the light of differentiation-dependent changes in the expression of individual cytokeratin polypeptides in developing epithelia.     

Cytokeratins in normal lung and lung carcinomas. I. Adenocarcinomas, squamous cell carcinomas and cultured cell lines

The various epithelial cells of the lower respiratory tract and the carcinomas derived from them differ markedly in their differentiation characteristics. Using immunofluorescence microscopy and two-dimensional gel electrophoresis of cytoskeletal proteins from microdissected tissues we have considered whether cytokeratin polypeptides can serve as markers of cell differentiation in epithelia from various parts of the human and bovine lower respiratory tract. In addition , we have compared these protein patterns with those found in the two commonest types of human lung carcinoma and in several cultured lung carcinoma cell lines. By immunofluorescence microscopy, broad spectrum antibodies to cytokeratins stain all epithelial cells of the respiratory tract, including basal, ciliated, goblet, and alveolar cells as well as all tumor cells of adenocarcinomas and squamous cell carcinomas. However, in contrast, selective cytokeratin antibodies reveal cell type-related differences. Basal cells of the bronchial epithelium react with antibodies raised against a specific epidermal keratin polypeptide but not with antibodies derived from cytokeratins characteristic of simple epithelia. When examined by two-dimensional gel electrophoresis, the alveolar cells of human lung show cytokeratin polypeptides typical of simple epithelia (nos. 7, 8, 18 and 19) whereas the bronchial epithelium expresses, in addition, basic cytokeratins (no. 5, small amounts of no. 6) as well as the acidic polypeptides nos. 15 and 17. Bovine alveolar cells also differ from cells of the tracheal epithelium by the absence of a basic cytokeratin polypeptide. All adenocarcinomas of the lung reveal a "simple-epithelium-type" cytokeratin pattern (nos. 7, 8, 18 and 19). In contrast, squamous cell carcinomas of the lung contain an unusual complexity of cytokeratins. We have consistently found polypeptides nos. 5, 6, 8, 13, 17, 18 and 19 and, in some cases, variable amounts of cytokeratins nos. 4, 14 and 15. Several established cell lines derived from human lung carcinomas (SK-LU-1, Calu -1, SK-MES-1 and A-549) show a uniform pattern of cytokeratin polypeptides (nos. 7, 8, 18 and 19), similar to that found in adenocarcinomas. In addition, vimentin filaments are produced in all the cell lines examined, except for SK-LU-1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Monoclonal antibodies to human cytokeratins: application to various epithelial and mesothelial cells

Immunohistological analysis of human tissue using monoclonal antibodies against cytokeratins, which are confined to cells of epithelial origin, is a valuable technique. Using human epidermal keratins as antigen, we prepared monoclonal antibodies against cytokeratins (ZK1, ZK7, ZK61 and ZK99) and against a desmosomal protein (ZK31). Immunohistochemical staining of human skin sections using these antibodies showed a specific reaction with the epidermis: ZK1 stained the entire epidermis, ZK7 only the basal layer, ZK61 and ZK99 the suprabasal layers, and ZK31 the cellular interfaces. In order to test for antibody specificity, immunoblots with human epidermal and amnion epithelial cytokeratin polypeptides, as well as immunofluorescence microscopy of simple epithelia (glandular and simple columnar epithelia) were performed. ZK1, ZK61 and ZK99 reacted preferentially with cytokeratin polypeptides of stratified squamous epithelia and ZK7 recognized cytokeratins of stratified and simple epithelia. When the ZK antibodies were tested on mesothelial cells in pleural effusions, only ZK7 reacted with these cells. Biochemical analysis of cytokeratin accumulation in cells of primary and long-term cultures indicated that the cytokeratin pattern of mesothelial cells was quite unstable, while that of amnion epithelial cells showed only minor quantitative changes. The use of these antibodies to determine the epithelial origin of cells present in pleural effusions is proposed.     

Diversity of cytokeratins. Differentiation specific expression of cytokeratin polypeptides in epithelial cells and tissues

Epithelial cells contain a cytoskeletal system of intermediate-sized (7 to 11 nm) filaments formed by proteins related to epidermal keratins (cytokeratins). Cytoskeletal proteins from different epithelial tissues (e.g. epidermis and basaliomas, cornea, tongue, esophagus, liver, intestine, uterus) of various species (man, cow, rat, mouse) as well as from diverse cultured epithelial cells have been analyzed by one and two-dimensional gel electrophoresis. Major cytokeratin polypeptides are identified by immunological cross-reaction and phosphorylated cytokeratins by [³²P]phosphate labeling in vivo.It is shown that different epithelia exhibit different patterns of cytokeratin polypeptides varying in molecular weights (range: 40,000 to 68,000) and electrical charges (isoelectric pH range: 5 to 8.5). Basic cytokeratins, which usually represent the largest cytokeratins in those cells in which they occur, have been found in all stratified squamous epithelia examined, and in a murine keratinocyte line (HEL) but not in hepatocytes and intestinal cells, and in most other cell cultures including HeLa cells. Cell type-specificity of cytokeratin patterns is much more pronounced than species diversity. Anatomically related epithelia can express similar patterns of cytokeratin polypeptides. Carcinomas and cultured epithelial cells often continue to synthesize cytokeratins characteristic of their tissue of origin but may also produce, in addition or alternatively, other cytokeratins. It is concluded: (1) unlike other types of intermediate-sized filaments, cytokeratin filaments are highly heterogeneous in composition and can contain basic polypeptides: (2) structurally indistinguishable filaments of the same class, i.e. cytokeratin filaments, are formed, in different epithelial cells of the same species, by different proteins of the cytokeratin family; (3) vertebrate genomes contain relatively large numbers of different cytokeratin genes which are expressed in programs characteristic of specific routes of epithelial differentiation; (4) individual cytokeratins provide tissue- or cell type-specific markers that are useful in the definition and identification of the relatedness or the origin of epithelial and carcinoma cells.     

Heterogeneity in the immunolocalization of cytokeratin specific monoclonal antibodies in the rat eye: evaluation of unusual epithelial tissue entities

Summary The immunocytochemical localization of cytokeratin and vimentin in rat eye tissues was investigated using a panel of 39 monoclonal antibodies specific for single or multiple of cytokeratin polypeptides and one polyclonal anti CK20 antiserum. The retinal and the ciliary body pigment epithelia only expressed cytokeratins 8 and 18, whereas the fetal retinal pigment epithelium and focally the adult epithelium, in the transition zone of retina and ciliary body, exhibited a reactivity for cytokeratin 19. In contrast, the non-pigmented ciliary epithelium was positive for vimentin only.In the rat conjunctiva distributed goblet cell clusters were selectively stained with cytokeratin 7, 8, 18 and 19 specific monoclonal antibodies. Among them a group of cytokeratin 8 and 18 specific monoclonal antibodies which stained the goblet cells as well as cytokeratin 8 and 18 positive internal controls did not react with either the cytokeratin 8 and 18 positive neuroectodermal cells of the rat eye nor the rat choroid plexus epithelium. This indicates differences in the phenotype e.g. conformational epitope changes, of neuroectodermal derived and other cytokeratins. The corneal and conjunctival epithelium showed a more complex distribution of squamous epithelium type cytokeratins. The limbal region as a transient zone connecting both epithelia exhibited a changing cytokeratin pattern. In general, the study emphasized the necessity to work with an enlarged antibody panel to avoid misleading results in the immunolocalization of cytokeratins.Dedicated to Prof. Dr. H.J. Scharf (Halle, FRG) on the occasion of his 70th birthday     

An unusual type of cytokeratin filament in cells of a human cloacogenic carcinoma derived from the anorectal transition zone

Epithelia-derived tumors (carcinomas) can be distinguished from mesenchymally derived tumors by the presence of intermediate-sized filaments of the cytokeratin type, which usually coincides with the absence of other types of intermediate-sized filaments such as vimentin filaments. In the course of diagnostic examinations of human tumors, using immunofluorescence microscopy, we have come across a case of an unusual carcinoma (Primary tumor and lymph node metastasis) positively stained not only with cytokeratin antibodies but also with immunoglobulins present in vimentin antisera. Therefore, this tumor, a cloacogenic carcinoma apparently derived from the rectal-anal transitional region, has been examined in greater detail using both immunofluorescence microscopy and immuno-electron microscopy as well as gel electrophoretic analysis of cytoskeletal polypeptides from total tumor tissue and from microdissected nodules enriched in carcinoma cells. The unusual reaction of the carcinoma cells with immunoglobulins present in seven different (rabbit or guinea pig) antisera raised against vimentin, has been found to be diminished after absorption on purified cytokeratin or total epidermal cytoskeletal material, but not after absorption on purified vimentin. Gel electrophoretic analysis of tumor cytoskeletons showed an unusual complex pattern of cytokeratin polypeptides containing relatively large (Mr 68,000 and Mr 58,000) neutral-to-slightly basic cytokeratins, as are typically found in epidermis and other stratified squamous epithelia, as well as several smaller acidic cytokeratins, including a Mr 40,000 polypeptide found in certain nonstratified epithelial such as colon and small intestine. Total tumor also showed the inclusion of some vimentin which, however, was significantly decreased in analysis of excised carcinoma nodules. Examining antibody binding to polypeptides separated by gel electrophoresis and blotted on nitrocellulose paper, we have found that antisera raised against vimentin contained not only vimentin antibodies but also immunoglobulins which specifically bound to the largest cytokeratin component. We conclude that the unusual reaction of immunoglobulins present in vimentin antisera with cytokeratin filament bundles does not represent specific binding to vimentin in these carcinoma cells, but is due to a component obviously widespread in vimentin antisera which binds specifically to a cytokeratin present in this type of tumor but not in most other carcinomas. It is proposed that use is made in diagnostic examinations of vimentin antisera or affinity-purified vimentin antibodies that have been pre-absorbed on cytokeratin protein, in order to eliminate such disturbing reactions.     

Extensive changes in cytokeratin expression patterns in pathologically affected human gingiva

The stratified squamous epithelium of the oral gingiva and the hard palate is characterized by a tissue architecture and a cytoskeletal composition similar to, although not identical with, that of the epidermis and fundamentally different from that of the adjacent non-masticatory oral mucosa. Using immunocytochemistry with antibodies specific for individual cytokeratins, in situ hybridization and Northern blots of RNA with riboprobes specific for individual cytokeratin mRNAs, and gel electrophoresis of cytoskeletal proteins of microdissected biopsy tissue samples, we show changes in the pattern of expression of cytokeratins and their corresponding mRNAs in pathologically altered oral gingiva. Besides a frequently, although not consistently, observed increase in the number of cells producing cytokeratins 4 and 13 (which are normally found as abundant components in the sulcular epithelium and the alveolar mucosa but not in the oral gingiva) and a reduction in the number of cells producing cytokeratins 1, 10 and 11, the most extensive change was noted for cytokeratin 19, a frequent cytokeratin in diverse one-layered and complex epithelia. While in normal oral gingiva cytokeratin 19 is restricted to certain, sparsely scattered cells of --or near--the basal cell layer, probably neuroendocrine (Merkel) cells, in altered tissue of inflamed samples it can appear in larger regions of the basal cell layer(s) and, in apparently more advanced stages, also in a variable number of suprabasal cells. Specifically, our in situ hybridization experiments show that this altered suprabasal cytokeratin 19 expression is more extended at the mRNA than at the protein level, indicating that cytokeratin 19 mRNA synthesis may be a relatively early event during the alteration. These changes in cytokeratin expression under an external pathological influence are discussed in relation to other factors known to contribute to the expression of certain cytokeratins and with respect to changes occurring during dysplasia and malignant transformation of oral epithelia.     

Changes in the Pattern of Cytokeratin Polypeptides in Epidermis and Hair Follicles During Skin Development in Human Fetuses

Abstract. The cytokeratin polypeptides of microdissected epidermis and hair follicles from human fetuses (from week 10 of pregnancy until birth) have been analysed by two-dimensional gel electrophoresis. Two-layered epidermis in 10-week fetuses contains major amounts of cytokeratin polypeptides typical of simple epithelia (components Nos. 8, 18, and 19 according to Moll et al. [31]). These cytokeratins are gradually reduced in their relative amounts and eventually disappear in the multilayered epidermis of later stages. At advanced stages of development, cytokeratins characteristic of adult epidermis are detected and finally predominate. These include the large and basic epidermal cytokeratin No. 1 (apparent molecular weight 68,000) which is already present in the three-layered epidermis of 13-week fetuses. Hair follicle germ cells of 13-week fetuses differ from fetal epidermal keratinocytes and show a very simple cytokeratin pattern, dominated by only two major polypeptides (Nos. 5 and 17). More developed hair follicles of 20-week fetuses have established a cytokeratin pattern similar to, but not identical with, that of hair follicles from adult skin. Different staining patterns obtained by indirect immunofluorescence microscopy using cytokeratin antibodies with different specificities suggest that, in three-layered epidermis, different cytokeratin patterns might exist in the specific cell layers. Such a differential location might explain the high complexity of polypeptide components found in fetal skin. Possible contributions of peridermal cytokeratins to this complex pattern of fetal epidermis are discussed.     

Cytokeratins in certain endothelial and smooth muscle cells of two taxonomically distant vertebrate species, Xenopus laevis and man

Using immunolocalization techniques, electron microscopy, and gel electrophoresis combined with immunoblotting, we have noted remarkable interspecies differences in the expression of cytokeratins in certain nonepithelial cells. In the present study we describe, in two taxonomically distant vertebrate species, the African clawed toad Xenopus laevis and man, endothelial and smooth muscle cells which express cytokeratin intermediate filaments (IFs), in addition to vimentin and/or desmin IFs. In Xenopus, all endothelia seem to produce both vimentin and cytokeratin IFs. As well, certain smooth muscle bundles located in the periphery of the walls of the esophagus and the urinary bladder produce small amounts of cytokeratin IFs in addition to IFs containing vimentin or desmin or both. The amphibian equivalents of human cytokeratins 8 and 18 have been identified in these nonepithelial tissues. In human endothelial cells, immunocytochemical reactions with certain cytokeratin antibodies are restricted to a rare subset of blood vessels. Vessels of this type were first noted in synovial and submucosal tissues, but also occur in some other locations. Cytokeratins have also been detected in certain groups of smooth muscles, such as those present in the walls of some blood vessels in synovial tissue and umbilical cord. Here, the synthesis of low levels of cytokeratins 8 and 18, sometimes with traces of cytokeratin 19, has been demonstrated in smooth muscle cells by colocalization with myogenic marker proteins, such as desmin and/or the smooth-muscle-specific isoform of alpha-actin. Possible reasons for the differences in cytokeratin expression between adjacent endothelia in man, and smooth-muscle structures in both species, as well as biologic and histodiagnostic implications of these findings, are discussed.     

Patterns of expression of trichocytic and epithelial cytokeratins in mammalian tissues II. Concomitant and mutually exclusive synthesis of trichocytic and epithelial cytokeratins in diverse human and bovine tissues (hair follicle, nail bed and matrix, lingual papilla, thymic reticulum)

The hair-forming cells (trichocytes) and the mature hair contain four major trichocytic cytokeratins from each of the subfamilies, basic (Hb1-4) and acidic (Ha1-4); these are related - but not identical - to the epithelial cytokeratins. Here we show, by biochemical methods and immunofluorescence microscopy using antibodies specific for either epithelial or trichocyte cytokeratins, that the same set of hair-type cytokeratins, including two newly identified minor components, designated Hax (type I) and Hbx (type II), are also expressed in cells forming nails, in the filiform papillae of the dorsal surface of human and bovine tongue, and, most surprisingly, in some cells of the epithelial reticulum of bovine and human thymus. By double-label immunofluorescence microscopy, we also show that the expression of the two subsets of cytokeratins, i.e., the epithelial and the trichocytic ones, is not necessarily mutually exclusive, but that certain cells of hair follicles, nail matrix and bed, lingual papillae, and the nonlymphoid cell system of the thymus contain both trichocytic and certain epithelial cytokeratins. This indicates that these cells coexpress representatives of both kinds of cytokeratin. Implications of these findings with respect to problems of regulatory control of cytokeratin synthesis in tissue development and differentiation, and the possible functional meaning of the occurrence of trichocytic cytokeratins in such histologically diverse tissues, are discussed.     

Cytokeratin polypeptide patterns of different epithelia of the human male urogenital tract: immunofluorescence and gel electrophoretic studies

Intermediate filament proteins of normal epithelia of the human and the bovine male urogenital tract and of certain human renal and bladder carcinomas have been studied by immunofluorescence microscopy and by two-dimensional gel electrophoresis of cytoskeletal fractions from microdissected tissue samples. The patterns of expression of cytokeratin polypeptides differ in the various epithelia. Filaments of a cytokeratin nature have been identified in all true epithelial cells of the male urogenital tract, including renal tubules and rete testis. Simple epithelia of renal tubules and collecting ducts of kidney, as well as rete testis, express only cytokeratin polypeptides nos. 7, 8, 18, and 19. In contrast, the transitional epithelia of renal pelvis, ureter, bladder, and proximal urethra contain, in addition to those polypeptides, cytokeratin no. 13 and small amounts of nos. 4 and 5. Most epithelia lining the human male reproductive tract, including those in the epididymis, ductus deferens, prostate gland, and seminal vesicle, synthesize cytokeratin no. 5 in addition to cytokeratins nos. 7, 8, 18, and 19 (cytokeratin no. 7 had not been detected in the prostate gland). Cytokeratin no. 17 has also been identified, but in very low amounts, in seminal vesicle and epididymis. The cytokeratin patterns of the urethra correspond to the gradual transition of the pseudostratified epithelium of the pars spongiosa (cytokeratins nos. 4, 5, 6, 13, 14, 15, and 19) to the stratified squamous epithelium of the fossa navicularis (cytokeratins nos. 5, 6, 10/11, 13, 15, and 19, and minor amounts of nos. 1 and 14). The noncornified stratified squamous epithelium of the glans penis synthesizes cytokeratin nos. 1, 5, 6, 10/11, 13, 14, 15, and 19. In immunofluorescence microscopy, selective cytokeratin antibodies reveal differential staining of different groups or layers of cells in several epithelia that may relate to the specific expression of cytokeratin polypeptides. Human renal cell carcinomas show a simple cytokeratin pattern consisting of cytokeratins nos. 8, 18, and 19, whereas transitional cell carcinomas of the bladder reveal additional cytokeratins such as nos. 5, 7, 13, and 17 in various proportions. The results shows that the wide spectrum of histological differentiation of the diverse epithelia present in the male urogenital tract is accompanied by pronounced changes in the expression of cytokeratin polypeptides and suggest that tumors from different regions of the urogenital tract may be distinguished by their cytokeratin complements.     

Modulation of intermediate-filament expression in developing cochlear epithelium

The present study was designed to characterize the expression and distribution of intermediate filaments (IFs) in the diverse cellular elements of inner-ear epithelium in guinea pig and man. Using immunofluorescence microscopy with a battery of IF-specific monoclonal antibodies, we show that the epithelium of the otocyst expresses cytokeratin (CK) polypeptides typical of simple epithelia. Cells in the early otic ganglion were also positively labelled for cytokeratins, suggesting that they are of otocystic epithelial origin. Cytokeratin distribution was largely homogeneous in the early cochlear duct as the epithelium differentiated, differences in the distribution of cytokeratin between the various cell types became detectable. Characteristically, cochlear hair cells became devoid of cytokeratin labelling, and remained unlabelled with antibodies specific for all other IF classes. The neural tissue of the inner ear was also devoid of cytokeratins and was typically positive for neurofilaments. Vimentin IFs were abundant in the mesenchymal tissues around the membranous labyrinth. Desmin and glial fibrillary acidic protein were not detectable in the cochlea. The apparent absence of all IFs from the cochlear hair cells in both guinea pig and man, as revealed by immunofluorescence and electron microscopy, and the possible significance of their absence for cochlear physiology, are discussed.     

Monoclonal antibodies to various acidic (type I) cytokeratins of stratified epithelia

We determined the reactivity of two monoclonal antibodies to cytokeratins that are typically expressed in certain stratified epithelia and several human squamous cell carcinomas using immunoblotting techniques and immunofluorescence microscopy. Antibody KS 8.12 reacted specifically with cytokeratin polypeptides nos. 13 and 16, and stained noncornified squamous epithelia in a rather uniform way. The examination of diverse human carcinomas showed all squamous cell carcinomas to be positively stained with this antibody, whereas all adenocarcinomas were negative. Another antibody, KK 8.60, reacted with polypeptides nos. 10 and 11, and uniformly stained the suprabasal layers of the epidermis. In several noncornified squamous epithelia (e.g., tongue, exocervix), in thymus reticulum epithelial cells, and in moderately and well differentiated squamous cell carcinomas this antibody exhibited a nonuniform labeling pattern that allowed the detection of individual cytokeratin-10/11-positive cells scattered throughout the tissue. It is concluded that antibodies KS 8.12 and KK 8.60 represent specific molecular probes for the definition of certain stages of squamous differentiation in normal development as well as in pathological processes such as squamous metaplasia and carcinogenesis. We propose the use of these antibodies in the differential diagnosis of carcinomas and their metastases.     

Cytokeratin filaments and desmonsomes in the epithelioid cells of the perineurial and arachnoidal sheaths of some vertebrate species

Using electron microscopy and immunohistochemistry with a large panel of antibodies to various cytoskeletal proteins we have noted that the single- or multi-layered sheaths of epithelioid cells ("neurothelia") surrounding peripheral nerves (perineurial cells) or structures of the central nervous system, including the optic nerve (arachnoid cells), show remarkable interspecies differences in their cytoskeletal complements. In two anuran amphibia examined (Xenopus laevis, Rana ridibunda), the cells of both forms of neurothelia, i.e., perineurial and arachnoid, are interconnected by true desmosomes and are rich intermediate-sized filaments (IFs) of the cytokeratin type. Among higher vertebrates, a similar situation is found in the bovine and chicken nervous systems, in which the arachnoid cells of the meninges contain desmosomes and IFs of both the cytokeratin (apparently with restricted epitope accessibilities in the chicken) and the vimentin type, whereas the perineurial cells of many nerves contain cytokeratin IFs, often together with vimentin, but no desmosomes. In contrast, in rat arachnoidal and perineurial cells significant reactions have been observed neither for cytokeratins nor for desmosomes. In the human nervous system, cytokeratins and desmosomes have also not been seen in the various perineuria studied whereas desmosomes are frequent in arachnoidal cell layers which are dominated by vimentin IFs and only in certain small regions of the brain contain some additional cytokeratins. The occurrence of cytokeratins in the tissues found positive by immunohistochemistry has been confirmed by gel electrophoresis of cytoskeletal proteins, followed by immunoblotting. Our results emphasize both similarities and differences between the neurothelia on the one hand and epithelia or endothelia on the other, justifying classification as a separate kind of tissue, i.e., neurothelium. The observations of interspecies differences lead to the challenging conclusion that neither desmosomes nor cytokeratins are essential for the basic functions of neurothelial sheaths nor does the specific type of IF protein expressed in these cells appear to matter in this respect. The results are also discussed in relation to the cytoskeletal characteristics of other epithelioid tissues and of human neurothelium-derived tumors.     

Patterns of expression of trichocytic and epithelial cytokeratins in mammalian tissues

Cells forming hair and nail material are characterized by the synthesis of members of a particular group of alpha-keratin polypeptides (trichocytic cytokeratins. "T cytokeratins") different from epithelial cytokeratins ("E cytokeratins"). As the precursor cells to trichocytes are derived from fetal epidermal keratinocytes expressing only E cytokeratins, we have studied the patterns of expression of both T and E cytokeratins in developing human hair-and nail-forming tissues of different fetal stages, by immunocytochemistry using antibodies specific for certain T or E cytokeratins and by two-dimensional gel electrophoresis and immunoblotting. In developing hair follicles up to the early bulbous-peg stage (weeks 12-15 of gestational age), only certain E but no T cytokeratins were identified. T cytokeratins were first detected in the late bulbous-peg stage (in week-14 scalp skin) in certain cells of the central part of the hair cone. In hair-producing follicles (weeks 18-25), the lower hair matrix cells were positive for certain E cytokeratins, whereas T cytokeratins appeared in the uppermost portion of the matrix and, most prominently, in the maturing trichocytes. From the late bulbous-peg stage on. E cytokeratin antibody Ks13.1 selectively decorated the inner root sheath. In finger nail "anlagen", T cytokeratins were detected first in week 12 and 13 fetuses, specifically in cells of the lunula region. In more-advanced stages of nail formation, expression of T cytokeratins extended not only to the upper layers of the ventral nail matrix but was also found, albeit more sparsely, in cells of the whole nail-bed epithelium. Throughout these developmental stages, coexpression of T and E cytokeratins was noted in certain cells, including E cytokeratin 19. While in earlier stages E cytokeratins 10/11, characteristic of epidermal-type cornification, were noted in different regions, including the superficial stratum of the nail bed epithelium, they were later restricted to the epithelium of the proximal nail fold. The results show that terminal trichocytic differentiation starts, both in ontogeny and during the steady growth of hairs and nails, in cells expressing E cytokeratins and that coexpression of E and T polypeptides occurs in both kinds of appendages. While in the hair follicle, the change to the exclusive synthesis of T cytokeratins appears to take place relatively abruptly and simply, the development of nail structures from the ventral nail matrix seems to be more gradual and is characterized by more-complex patterns of coexpression of both kinds of cytokeratins.     

Cytokeratin expression in squamous metaplasia of the human uterine cervix

The expression of cytokeratin polypeptides in squamous metaplasia of the human uterine cervix was investigated by immunocytochemical labeling with polypeptide-specific antibodies against cytokeratins. Immunofluorescence microscopic examination of cervical tissues using various monoclonal antibodies indicated that squamous cervical metaplasia expresses a unique set of cytokeratin polypeptides, this being distinctively different from that expressed by all of the normal epithelial elements of the exo- and endocervix. The development of metaplastic foci was accompanied by the expression of cytokeratin polypeptide no. 13, which is commonly detected in stratified epithelia, and by a reduction in the level of polypeptide no. 18, which is typical of simple epithelia. The 40-kilodalton cytokeratin (no. 19) described by Moll et al., which is abundant in the columnar and reserve cells of the endocervix, was found throughout the metaplastic lesions. Only in 'well-differentiated' metaplasias did we detect polarity of cytokeratin expression reminiscent of the staining patterns in the exocervix. This was manifested by the exclusive labeling of the basal cell layer(s) with antibodies KB 8.37 and KM 4.62, which stain the basal cells of the exocervix. Furthermore, a comparison of cervical metaplasia with squamous areas occurring within endometrial adenocarcinomas pointed to a close similarity in the cytokeratin expression of the two. We discuss the use of cytokeratins as specific markers of squamous differentiation, the relationships between squamous metaplasia and cervical neoplasia, and the involvement of reserve cells in the metaplastic process.     

Molecular characterization and expression of the stratification-related cytokeratins 4 and 15

A number of human cytokeratins are expressed during the development of stratified epithelia from one-layered polar epithelia and continue to be expressed in several adult epithelial tissues. For studies of the regulation of the synthesis of stratification-related cytokeratins in internal tissues, we have prepared cDNA and genomic clones encoding cytokeratin 4, as a representative of the basic (type II) cytokeratin subfamily and cytokeratin 15, as representative of the acidic (type I) subfamily, and determined their nucleotide sequences. The specific expression of mRNAs encoding these two polypeptides in certain stratified tissues and cultured cell lines is demonstrated by Northern blot hybridization. Hybridization in situ with antisense riboprobes and/or synthetic oligonucleotides shows the presence of cytokeratin 15 mRNA in all layers of esophagus, whereas cytokeratin 4 mRNA tends to be suprabasally enriched, although to degrees varying in different regions. We conclude that the expression of the genes encoding these stratification-related cytokeratins starts already in the basal cell layer and does not depend on vertical differentiation and detachment from the basal lamina. Our results also show that simple epithelial and stratification-related cytokeratins can be coexpressed in basal cell layers of certain stratified epithelia such as esophagus. Implications of these findings for epithelial differentiation and the formation of squamous cell carcinomas are discussed.     

Intermediate filament cytoskeleton of amnion epithelium and cultured amnion epithelial cells: expression of epidermal cytokeratins in cells of a simple epithelium

Using immunofluorescence microscopy and two-dimensional gel electrophoresis, we compared the cytoskeletal proteins expressed by human amnion epithelium in situ, obtained from pregnancies of from 10-wk to birth, with the corresponding proteins from cultured amnion epithelial cells and cultures of cells from the amniotic fluid of 16 week pregnancies. Epithelia of week 16 fetuses already display tissue-specific patterns of cytokeratin polypeptides which are similar, although not identical, to those of the corresponding adult tissues. In the case of the simple amnion epithelium, a complex and characteristic complement of cytokeratin polypeptides of Mr 58,000 (No. 5), 56,000 (No. 6), 54,000 (No. 7), 52,500 (No. 8), 50,000 (No. 14), 46,000 (No. 17), 45,000 (No. 18), and 40,000 (No. 19) is present by week 10 of pregnancy and is essentially maintained until birth, with the addition of cytokeratin No. 4 (Mr 59,000) and the disappearance of No. 7 (Mr 54,000) at week 16 of pregnancy. In full-term placentae, the amnion epithelium displays two morphologically distinct regions, i.e., a simple and a stratified epithelium, both of which express the typical amnion cytokeratin polypeptides. However, in addition the stratified epithelium also synthesizes large amounts of special epidermal cytokeratins such as No. 1 (Mr 68,000), 10 (Mr 56,500), and 11 (Mr 56,000). In culture amnion epithelial cells obtained from either 16-wk pregnancies or full-term placentae will continue to synthesize the amnion-typical cytokeratin pattern, except for a loss of detection of component No. 4. This pattern is considerably different from the cytokeratins synthesized by cultures of cells from amniotic fluids (cytokeratins No. 7, 8, 18, and 19, sometimes with trace amounts of No. 17) and from several so-called "amnion epithelial cell lines." In addition, amnion epithelial cells in situ as well as amnion epithelial cell cultures appear to be heterogeneous in that they possess some cells that co-express cytokeratins and vimentin. These observations lead to several important conclusions: In contrast to the general concept of recent literature, positively charged cytokeratins of the group No. 4-6 can be synthesized in a simple, i.e., one-layered epithelium. The change from simple to stratified amnion epithelium does not require a cessation of synthesis of cytokeratins of the simple epithelium type, but in this case keratins characteristic of the terminally differentiated epidermis (No. 1, 10, and 11) are also synthesized.(ABSTRACT TRUNCATED AT 400 WORDS)