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Twelve cases of gastrointestinal neuroendocrine tumors, including eight carcinoids and four pancreatic islet cell tumors or their metastases, were immunohistochemically analyzed for the expression of different types of intermediate filament proteins. All of the tumors showed cytokeratin positivity in immunostaining, and the Western blotting technique revealed 45- and 52-kilodalton cytokeratins in carcinoid tumors. Three of the islet cell tumors, but none of the carcinoid tumors, showed, in addition, varying numbers of neurofilament-positive tumor cells when evaluated with rabbit and mouse monoclonal antineurofilament antibodies. The presence of only the 70-kilodalton neurofilament and cytokeratin polypeptides in an islet cell tumor was revealed also by using the Western blotting technique. On the other hand, both fetal and adult pancreatic islet cells showed only cytokeratin positivity. Neurofilament-positive epithelial cells were not found in normal small intestines either. The results show epithelial characteristics in normal gastrointestinal neuroendocrine cells and neuroendocrine tumors by their expression of cytokeratin. In addition, some islet cell tumors display the 70-kilodalton neurofilament protein which suggests the acquisition of a new type of intermediate filament during the neoplastic change.

The effects of epidermal growth factor (EGF) on the cytokeratin filaments of cultured murine epithelial cells were studied by the indirect immunofluorescence technique with affinity-purified antibodies. Mouse epithelial cells (MMC-E), grown on glass cover slips, and viewed by immunofluorescence microscopy, showed keratin-specific fluorescence as typical bright perinuclear aggregates corresponding to dense paracrystalline granules seen in electron microscopy. Within minutes after an exposure to EGF, the keratin granules in the MMC-E cells decreased. After 10 min of incubation, the cells had spread fibrillar keratin. Such an effect could not be found after a similar exposure to insulin, dexamethasone, dibutyryl cyclic AMP, or antimitotic drugs. EGF, therefore, has a relatively direct effect on the cytoskeletal organization of cultured epithelial cells. These rapid effects on the keratin filaments may explain the simultaneous EGF-induced ultrastructural surface changes of the cells. EGF may thus function as a regulatory factor in the migration of epithelial cells and in the mobility of their cell membranes. The epithelial cell line, MMC-E, should prove a useful model for studies on the action of EGF on nontransformed epithelial cells in vitro.

Four cases of classical biphasic synovial sarcoma were studied for intermediate filaments of keratin and vimentin type. Epithelial-like cells lining gland-like slits were strongly positive for keratin but negative for vimentin, whereas the spindle cell stroma was negative for keratin but positive for vimentin. The observations indicate epithelial differentiation in the glandular elements of biphasic synovial sarcomas, and are consistent with earlier ultrastructural observations suggesting epithelial properties of these cells.

Expression of cytokeratins and vimentin in developing and adult human kidney was studied by the indirect immunofluorescence technique using monoclonal and conventional antiintermediate filament antibodies. The undifferentiated cells of the metanephric mesenchyme expressed vimentin but not cytokeratins, whereas only cytokeratins could be visualized in the cells of induced renal vesicles. At the S-shaped body stage of nephrogenesis, the presumptive visceral and parietal cells of the developing glomeruli did not contain either detectable vimentin or cytokeratins, whereas cells of the tubular pole of the S-shaped body were stained within antibodies to cytokeratins. At a later stage the cuboidal visceral epithelial cells of the presumptive glomeruli were brightly positive for vimentin, but not for cytokeratins, and maintained their expression of vimentin during later stages of glomerulogenesis and in adult kidneys. Only some of the cells of the parietal epithelium, but not other glomerular elements, showed cytokeratin-specific staining. In fetal kidneys, the epithelial cells of proximal and distal tubules and of collecting ducts showed a cytoplasmic staining with two monoclonal antibodies to cytokeratins (PKK1 and PKK2), reacting with different cytokeratin polypeptides. In adult kidneys, a basolateral fluorescence in proximal and distal tubules was obtained with PKK1 antibodies, whereas PKK2 stained only the cells of the collecting ducts. Vimentin was not found in tubular epithelial cells at any developmental stage, whereas the cells of collecting ducts showed a transient expression of vimentin in fetal kidneys. Our results show a developmental stage-dependent pattern in the expression of the intermediate filament antigens in the cells of the kidney and show the exceptional expression of only vimentin in the visceral epithelial cells of human glomeruli.

The presence and distribution of intermediate filament proteins in mouse oocytes and preimplantation embryos was studied. In immunoblotting analysis of electrophoretically separated polypeptides, a distinct doublet of polypeptides with Mr of 54K and 57K, reactive with cytokeratin antibodies, was detected in oocytes and in cleavage-stage embryos. A similar doublet of polypeptides, reactive with cytokeratin antibodies, was also detected in late morula-and blastocyst-stage embryos, and in a mouse embryo epithelial cell line (MMC-E). A third polypeptide with Mr of 50K, present in oocytes only as a minor component, was additionally detected in the blastocyst-stage embryos. No cytokeratin polypeptides could be detected in granulosa cells. Immunoblotting with vimentin antibodies gave negative results in both cleavage-stage and blastocyst-stage embryos. In electron microscopy, scattered filaments, 10-11 nm in diameter, were seen in detergent-extracted cleavage-stage embryos. Abundant 10-nm filaments were present in the blastocyst outgrowth cells. In indirect immunofluorescence microscopy (IIF) of oocytes and cleavage-stage embryos, diffuse cytoplasmic staining was seen with antibodies to cytokeratin polypeptides but not with antibodies to vimentin, glial fibrillary acidic protein, or neurofilament protein. Similarly, the inner cell mass (ICM) cells in blastocyst outgrowths showed diffuse cytokeratin-specific fluorescence. We could not detect any significant fibrillar staining in cleavage-stage cells or ICM cells by the IIF method. The first outgrowing trophectoderm cells already had a strong fibrillar cytokeratin organization. These immunoblotting and -fluorescence results suggest that cytokeratin-like polypeptides are present in mouse oocytes and preimplantation-stage embryos, and the electron microscopy observations show that these early stages also contain detergent-resistant 10- to 11-nm filaments. The relative scarcity of these filaments, as compared to the high intensity in the immunoblotting and immunofluorescence stainings, speaks in favor of a nonfilamentous pool of cytokeratin in oocytes and cleavage-stage embryos.

Previous studies have suggested that cytoskeletal elements in an altered form in vivo (alcoholic hyalin , AH) are targets for an autoimmune reaction in alcoholic liver disease (ALD). In this study we assayed autoantibodies to cytoskeletal cytoskeletal cytokeratin filaments, intermediate filaments of epithelial cells, by indirect immunofluorescence technique (IIF) using cultured human amnion epithelial cells and PtK 2 cells as targets. Sera from 20 patients with ALD, 38 patients with urogenital cancer, 22 patients with renal diseases, and from 18 healthy controls were studied. Patients with ALD had a significantly increased prevalence of cytokeratin filament autoantibodies using both PtK 2 cells (69%) and human amnion epithelial cells (69%) as substrate, as compared to other groups. The antibodies were mainly of IgA and IgM class and could be demonstrated in all forms of ALD. Interestingly, cytoskeleton antibodies have previously been observed in diseases of viral and "autoimmune" origin.

Monophasic synovial sarcomas of spindle-cell type and fibrosarcomas were studied by electron and immunofluorescence microscopy for their intermediate filament expression and the binding of peanut agglutinin (PNA). In monophasic synovial sarcomas of spindle-cell type (two cases), frequent cell-to-cell junctions, irregular cytoplasmic processes, and occasional cytoplasmic, tonofilament-like bundles of intermediate filaments were seen by electron microscopy. These features were absent from fibrosarcomas. Immunohistologically, the monophasic synovial sarcomas showed arrays of prekeratin-positive cells in the midst of the vimentin-positive spindle cells. By double fluorescence microscopy, the prekeratin-positive cells also bound PNA, like the epithelial-like cells of the classical biphasic synovial sarcoma. In contrast to monophasic synovial sarcomas, prekeratin-positive cells and arrays of PNA-binding cells, were not seen by immunofluorescence microscopy in fibrosarcomas (seven cases). Thus the prekeratin-content, the binding of PNA lectin, and certain ultrastructural features suggesting early epithelial differentiation, help to distinguish monophasic synovial sarcomas of spindle-cell type from other spindle cell sarcomas.

The expression of intermediate filaments of the keratin- and the vimentin-type was studied in heterokaryons of human fibroblasts and amnion epithelial cells by immunofluorescence microscopy. Fibroblasts and their homokaryons showed a fibrillar, vimentin-specific fluorescence throughout the cytoplasm but were negative when stained for keratin. Amnion epithelial cells and their homokaryons, on the other hand, showed a keratin-specific fibrillar staining, and only some of them contained also detectable vimentin. When suspended epithelial cells were fused with adherent fibroblasts, keratin fibrils spread within 3 h into the fibroblasts, intermixing with the vimentin fibrils. 1-3 d after fusion, both vimentin and keratin filaments were expressed as typical fibrillar cytoplasmic arrays, and the distribution of keratin in heterokaryons resembled closely that of vimentin. A typical cell-to-cell arrangement of keratin fibrils, seen in cultures of amnion epithelial cells, could also be found between heterokaryons. Treatment of the cultures with vinblastine sulphate induced coiling of the vimentin filaments in both homo- and heterokaryons, whereas the keratin organization was only slightly affected. Our results show that both vimentin and keratin filaments are incorporated into the cytoskeleton of heterokaryons formed between fibroblasts and epithelial cells, and that they behave in the same way as in their parental cells. Both epithelial and fibroblastic characteristics thus appear to the coexpressed in such heterokaryons.

The expression of cytokeratins in Paget cells in mammary and extramammary Paget's disease was studied using different keratin antibodies and immunofluorescence microscopy. Antibodies to epidermal keratin did not react with the Paget cells but stained the surrounding epidermis. Two monoclonal cytokeratin antibodies (PKKI and RGE 53), which reacted typically with simple glandular epithelia in normal tissues, brightly stained the Paget cells and left the surrounding epidermal cells unstained. The results indicate that Paget cells are derived from mammary or sweat duct epithelium rather than from epidermal cells.

Eight cases of primary and metastatic nasopharyngeal lymphoepithelioma and four cases of malignant lymphoma of the pharyngeal region were studied for the presence of keratin by indirect immunofluorescence microscopy. The results showed that all the cases of primary and metastatic lymphoepithelioma contained keratin-positive cells, whereas all the lymphomas were negative for keratin. Anti-keratin antibody thus seems to be a valuable aid in the differential diagnosis between lymphoepithelioma and lymphoma.

The expression of cytokeratin and vimentin type intermediate filaments were studied in fetal, postnatal, and adult rat testes. Immunocytochemical observations were correlated with the light and electron microscopic analysis of the developing organs. The Sertoli cell precursors in 15-day-old fetal testes contained both cytokeratin and vimentin. A gradual reorganization of both filaments, accompanied by a decrease of cytokeratin-positivity, was observed toward the end of the fetal period. The simultaneous presence of cytokeratin and vimentin in the same cells was shown by double immunofluorescence of newborn testes and the primary culture of dissociated testicular cells. In postnatal Sertoli cells, cytokeratin-positivity continued to decrease and disappeared by the age of 14 days. The increase in vimentin content and the appearance of axially oriented vimentin filaments coincided with the acquisition of the columnar shape of the Sertoli cells. The presence of cytokeratin and vimentin in fetal and newborn testes, and only vimentin in the adult testes was confirmed by immunoblotting. The present results suggest that major qualitative changes in the expression of intermediate filament proteins can take place during the embryonic development. The expression of cytokeratin in developing Sertoli cells, although only transient, supports the epithelial origin of these cells and can be applied as a marker for embryonic and early postnatal Sertoli cells.

Intermediate filaments (IF) are ubiquitous cytoplasmic structures which, by virtue of their cell- and tissue-type-specific characteristics, are widely used as markers of tissue derivation and as differential diagnostic aids in surgical pathology. In contradistinction to other IFs, vimentin filaments, characteristic of mesenchymal cells, may be co-expressed with other cell-type specific IFs--cytokeratin filaments, desmin filaments, glial filaments and neurofilaments--in some tumor cells, embryonic cells, and cells in vitro. In this study we describe a novel type of IF co-expression which does not involve vimentin-filaments, viz. the presence of both cytokeratin filaments and neurofilaments in human bronchial carcinoid tumor cells.