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)     

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.     

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.     

Cytokeratin expression in human thymus: immunohistochemical mapping

Cytokeratin expression in normal postnatal human thymus was studied immunohistochemically by using monoclonal antibodies against various cytokeratin polypeptides. An attempt was made to characterize cell populations giving rise to the cornified structures of Hassal's corpuscles. Monoclonal antibody KB-37, a marker of squamous epithelium basal cells, was applied to distinguish the earliest cells capable of undergoing squamous differentiation. Parts of the subcapsular epithelium were extensively stained with this reagent. This epithelium, like the basal layer of certain squamous epithelia, exibited a high incidence of cytokeratins 13 and 14, and pronounced expression of cytokeratin 19. Simple epithelium cytokeratins 8, 18, and 19 were present in the cortex. Scattered cells reacted with KB-37 antibody. All stellate epithelial cells in the medulla were positive for cytokeratin 19. Most of the medullar epithelial cells were positive for cytokeratins 13, 14 and 17 of complex epithelium, in contrast to the cortex, where only a few cells were positive for these cytokeratins. A significant proportion of the medullar cells was positive for KB-37 antigen. Cytokeratins 8 and 18 were expressed in single cells and in groups of cells surrounding Hassal's corpuscles. The outermost cells of these corpuscles were positive for cytokeratin 19 and KB-37. In the peripheral parts of Hassal's corpuscles, simple epithelium cytokeratins 7, 8, 18, and cytokeratins 4, 13, 14, and 17, characteristic of stratified nonkeratinizing epithelia, were coexpressed with keratinization-specific cytokeratins 10/11. The inner parts of the swirls were uniformly positive for cytokeratins was reduced.     

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)     

Cytoskeletal proteins as tissue-specific markers in cytopathology

Antibodies to intermediate filament proteins react in a tissue-specific manner and can be used to characterize tumor cells present in thin-needle aspirates from solid tumors, from palpable lymph nodes and cells present in samples from peritoneal and pleural effusions. From our studies so far the following conclusions can be drawn: Polyclonal antisera to cytokeratins can identify carcinoma metastases in thin-needle aspirates from palpable lymph nodes and distinguish them from malignant lymphomas and nonmalignant lesions such as chronic lymphadenitis, which show only vimentin-positive cells. Monoclonal antibodies to specific cytokeratin polypeptides are able to distinguish between different types of epithelial tumor metastases, i.e. metastases from adenocarcinomas and metastases from squamous cell carcinomas. Cells present in peritoneal and pleural effusions can be partly characterized using intermediate filament antisera. We have found that metastatic adenocarcinoma cells from breast, ovary, endometrium, cervix, colon and stomach, as well as squamous cell carcinomas and malignant mesothelioma stain specifically with antibodies to cytokeratin while mesenchymally derived tumors such as malignant lymphomas, malignant melanomas, and fibrosarcomas, are positive for vimentin only. Metastatic tumor cells of epithelial origin present in aspirates from human serous body cavity fluids may coexpress vimentin next to their original cytokeratin intermediate filaments. Benign mesothelial cells present in body cavity fluids frequently coexpress cytokeratins and vimentin. Tumor cells present in thin-needle aspirates from solid tumors such as pleomorphic adenomas of the parotid gland can be identified as such because of their typical patterns of intermediate filament (co-)expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

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.     

Distribution of intermediate-filament proteins in the human enamel organ: unusually complex pattern of coexpression of cytokeratin polypeptides and vimentin

We applied immunohistochemical techniques and gel electrophoresis to examine the distribution of intermediate filaments in human fetal oral epithelium and the epithelia of the human enamel organ. Both methods demonstrated that human enamel epithelia contain cytokeratins 5, 14, and 17, which are typical of the basal cells of stratified epithelia, as well as smaller quantities of cytokeratins 7, 8, 19, and in trace amounts 18, which are characteristic components of simple epithelial cells. In the external enamel epithelium and stellate-reticulum cells, most of these components appeared to be simultaneously expressed. In contrast, the parental oral epithelium was negative for cytokeratin 7, thus indicating possible "neoexpression" during the course of tooth formation. Immunohistochemical procedures using various monoclonal antibodies against vimentin revealed the transient coexpression of vimentin and cytokeratins in the external enamel epithelium and in stellate-reticulum cells during enamel development. The significance of the coexpression of cytokeratins and vimentin is discussed in relation to previous findings obtained in other normal tissues and in the light of the functional processes characteristic of these epithelia.     

Characterization of subcolumnar reserve cells and other epithelia of human uterine cervix. Demonstration of diverse cytokeratin polypeptides in reserve cells

We have analyzed the expression of cytokeratin polypeptides in subcolumnar reserve cells of the human uterine endocervical mucosa and the other epithelial cells using immunoperoxidase and immunofluorescence microscopy as well as by applying two-dimensional gel electrophoresis to microdissected cytoskeletal preparations. Endocervical columnar cells were uniformly positive for antibodies directed against the simple epithelium-type cytokeratins nos. 7, 8, 18, and 19, while a variable proportion of these cells was stained by an antibody against cytokeratin no. 4. Reserve cells were not only positive for cytokeratins nos. 8 (weakly and variably) and 19 but were also decorated by antibody KA 1, which reacts with cytokeratins present in stratified squamous epithelia. This last antibody selectively decorated reserve cells even when they were flat and inconspicuous. Antibody KA 1 uniformly stained the ectocervical squamous epithelium, the basal cells of which were also decorated by antibodies directed against cytokeratins nos. 8 (weakly and variably) and 19. Ectocervical suprabasal cells were positive, to a variable extent, for antibodies against cytokeratins nos. 4, 10/11, and 13. Gel electrophoresis revealed the presence of squamous-type cytokeratins nos. 5 and 17 in reserve cell-rich, but not in reserve cell-free, endocervical mucosa. We also analyzed the distribution pattern of these cells, as revealed by antibody KA 1, in the endocervical mucosa of 26 uteri. In all the specimens examined reserve cells were present, but their numbers exhibited considerable variation. In some cases these cells were confined to small islets localized deep within the cervical canal and lacked any continuity with the squamous epithelium. The expression of cytokeratins nos. 5 and 17 in reserve cells indicates that these cells have undergone a low level of squamous differentiation. The additional expression of cytokeratins nos. 8 and 19 in these cells points to a relationship with simple epithelial cells. The present data would seem to favor the view that reserve cells originate in situ from the columnar epithelium; however, this would imply an acquisition of new differentiation properties.     

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 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.     

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.     

Detection of a cytokeratin determinant common to diverse epithelial cells by a broadly cross-reacting monoclonal antibody.

A monoclonal antibody derived from a mouse immunized with bovine epidermal prekeratin has been characterized by its binding to cytoskeletal polypeptides separated by one- or two-dimensional gel electrophoresis and by immunofluorescence microscopy. This antibody (KG 8.13) binds to a determinant present in a large number of human cytokeratin polypeptides, notably some polypeptides (Nos. 1, 5, 6, 7, and 8) of the 'basic cytokeratin subfamily' defined by peptide mapping, as well as a few acidic cytokeratins such as the epidermis-specific cytokeratins Nos. 10 and 11 and the more widespread cytokeratin No. 18. This antibody reacts specifically with a wide variety of epithelial tissues and cultured epithelial cells, in agreement with previous findings that at least one polypeptide of the basic cytokeratin subfamily is present in all normal and neoplastic epithelial cells so far examined. The antibody also reacts with corresponding cytokeratin polypeptides in a broad range of species including man, cow, chick, and amphibia but shows only limited reactivity with only a few rodent cytokeratins. The value of this broad-range monoclonal antibody, which apparently recognizes a stable cytokeratin determinant ubiquitous in human epithelia, for the immunohistochemical identification of epithelia and carcinomas is discussed.     

A subfamily of relatively large and basic cytokeratin polypeptides as defined by peptide mapping is represented by one or several polypeptides in epithelial cells

Epithelial cells contain a class of intermediate-sized filaments formed by proteins related to epidermal alpha-keratins ('cytokeratins'). Different epithelia can express different combinations of cytokeratin polypeptides widely varying in apparent mol. wt. (40 000-68 000) and isoelectric pH (5.0-8.5). We have separated, by two-dimensional gel electrophoresis, cytokeratin polypeptides from various tissues and cultured cells of man, cow, and rodents and examined their relatedness by tryptic peptide mapping. By this method, a subfamily of closely related cytokeratin polypeptides has been identified which comprises the relatively large (greater than or equal to mol. wt. 52 500 in human cells) and basic (pH greater than or equal to 6.0) polypeptides but not the smaller and acidic cytokeratins. In all species examined, the smallest polypeptide of this subfamily is cytokeratin A, which is widespread in many simple epithelia and is the first cytokeratin expressed during embryogenesis. This cytokeratin polypeptide subfamily is represented by at least one member in all epithelial and carcinoma cells examined, indicating that polypeptides of this subfamily serve an important role as tonofilament constitutents . Diverse stratified epithelia and tumours derived therefrom contain two or more polypeptides of this subfamily, and the patterns of expression in different cell types suggest that some polypeptides of this subfamily are specific for certain routes of epithelial differentiation.     

Expression of simple epithelial type cytokeratins in stratified epithelia as detected by immunolocalization and hybridization in situ

Multi-layered ("stratified") epithelia differ from one-layered ("simple") polar epithelia by various architectural and functional properties as well as by their cytoskeletal complements, notably a set of cytokeratins characteristic of stratified tissue. The simple epithelial cytokeratins 8 and 18 have so far not been detected in any stratified epithelium. Using specific monoclonal antibodies we have noted, in several but not all samples of stratified epithelia, including esophagus, tongue, exocervix, and vagina, positive immunocytochemical reactions for cytokeratins 8, 18, and 19 which in some regions were selective for the basal cell layer(s) but extended into suprabasal layers in others. In situ hybridization with different probes (riboprobes, synthetic oligonucleotides) for mRNAs of cytokeratin 8 on esophageal epithelium has shown, in extended regions, relatively strong reactivity for cytokeratin 8 mRNA in the basal cell layer. In contrast, probes to cytokeratin 18 have shown much weaker hybridization which, however, was rather evenly spread over basal and suprabasal strata. These results, which emphasize the importance of in situ hybridization in studies of gene expression in complex tissues, show that the genes encoding simple epithelial cytokeratins can be expressed in stratified epithelia. This suggests that continual expression of genes coding for simple epithelial cytokeratins is compatible with the formation of squamous stratified tissues and can occur, at least in basal cell layers, simultaneously with the synthesis of certain stratification-related cytokeratins. We also emphasize differences of expression and immunoreactivity of these cytokeratins between different samples and in different regions of the same stratified epithelium and discuss the results in relation to changes of cytokeratin expression during fetal development of stratified epithelia, in response to environmental factors and during the formation of squamous cell carcinomas.     

Patterns of expression of trichocytic and epithelial cytokeratins in mammalian tissues. I. Human and bovine hair follicles

The cytokeratin family of intermediate filament (IF) proteins can be grouped into the epithelial polypeptides ("soft alpha-keratins"), of which at least 19 exist in the various human epithelia, and the hair-type cytokeratins ("hard alpha-keratins"), which are typical of trichocytes, i.e., the living hair-forming cells. We have recently shown [34] that the hair follicles from diverse mammalian species contain a set of eight major cytokeratin polypeptides, four each of the acidic (type I) and the basic (type II) subfamily, which are different from all known epithelial cytokeratins. In addition, we have identified two new minor trichocytic cytokeratin polypeptides, designated Hax (type I) and Hbx (type II). Antibodies against trichocytic cytokeratins that do not crossreact with any of the epithelial cytokeratins have enabled us to study the expression of both kinds of cytokeratin in the various cell types of human and bovine hair follicles. Using immunofluorescence microscopy, we have observed intense reactions of trichocytic cytokeratins only in cells contributing to the forming hairs, i.e., hair shaft, medulla and cuticle, whereas immunostaining of the peribulbar matrix cells was weaker, if at all detectable. In contrast, epithelial cytokeratins were localized in both the inner and outer root sheath epithelia but, surprisingly, also in certain portions of the trichocyte column, notably cells of the cuticle, certain medullary cells, and trichocytes of the basalmost peripapillary cell layers. Cells coexpressing trichocytic and epithelial cytokeratins have been identified by double-label immunofluorescence microscopy. Epithelial cytokeratins of the inner and outer root sheath epithelia include, most remarkably, "simple-epithelium-type" cytokeratins 8, 18, and 19; these occur in certain peribulbar regions, in distinct patterns, but with variable frequencies. The occurrence of simple epithelial cytokeratins in hair follicles has also been confirmed by high-sensitivity immunoblotting of follicular polypeptides separated by gel electrophoresis. Vimentin-positive cells were abundantly interspersed (in some follicles, but not in all) between the trichocytes of the peripapillary cone, most of them probably being melanocytes. The cell-type complexity of the hair follicle and the different patterns of cytoskeletal protein expression in the various hair follicle cells are discussed in relation to the development and growth of this organ.     

Different keratin polypeptides in epidermis and other epithelia of human skin: a specific cytokeratin of molecular weight 46,000 in epithelia of the pilosebaceous tract and basal cell epitheliomas

Cytokeratin polypeptides of human epidermis, of epithelia microdissected from various zones of the pilosebaceous tract (outer root-sheath of hair follicle, sebaceous gland), and of eccrine sweat-glands have been separated by one- and two-dimensional gel electrophoresis and characterized by binding of cytokeratin antibodies and by peptide mapping. The epithelium of the pilosebaceous tract has three major keratin polypeptides in common with interfollicular epidermis (two basic components of mol wts 58,000 and 56,000 and one acidic polypeptide of mol wt 50,000); however, it lacks basic keratin polypeptides in the mol wt range of 64,000-68,000 and two acidic keratin-polypeptides of mol wts 56,000 and 56,500 and contains an additional characteristic acidic cytokeratin of mol wt 46,000. Another cytokeratin polypeptide of mol wt 48,000 that is prominent in hair-follicle epithelium is also found in nonfollicular epidermis of foot sole. Both epidermis and pilosebaceous tract are different from eccrine sweat-gland epithelium, which also contains two major cytokeratins of mol wts 52,500 and 54,000 (isoelectric at pH 5.8-6.1) and a more acidic cytokeratin of mol wt 40,000. A striking similarity between the cytokeratins of human basal-cell epitheliomas and those of the pilosebaceous tract has been found: all three major cytokeratins (mol wts 58,000; 50,000; 46,000) of the tumor cells are also expressed in hair-follicle epithelium. The cytokeratin of mol wt 46,000, which is the most prominent acidic cytokeratin in this tumor, is related, by immunological and peptide map criteria, to the acidic keratin-polypeptides of mol wts 48,000 and 50,000, but represents a distinct keratin that is also found in other human tumor cells such as in solid adamantinomas and in cultured HeLa cells. The results show that the various epithelia present in skin, albeit in physical and ontogenic continuity, can be distinguished by their specific cytokeratin-polypeptide patterns and that the cytoskeleton of basal-cell epitheliomas is related to that of cells of the pilosebaceous tract.