Veli-Pekka Lehto

Diagnostic Application of Monoclonal Antibodies to Intermediate Filamentsa

Most of the histopathologic practice in pathology laboratories is based on histochemical staining techniques that reveal the major components common to most tissues or cells, or on more specific staining reactions, that reveal structural components or products typical of certain tissues or cells. The assortment of the utilizable specific stains is, however, narrow and therefore the correct identification of the specific nature of a given lesion is critically dependent on the personal experience of the pathologist. In order to improve the accuracy and reliability of the histopathological diagnostics, much effort has been devoted to develop new and more specific techniques that could be used to reveal specific compositional features of cells and tissues. The most successful approach has been the use of antibodies-either conventional polyclonal or monoclonal antibodies raised against specific tissue components that, combined with immunohistochemical techniques, can be used to retrieve more specific information on the compositional features of a lesion, e.g. tumor, which may allow its identification. Such new reagents include, for instance, antibodies that enable us to identify specific cell types in tissue sections, such as endothelial cells, histiomonocytic cells, different types of lymphatic cells, epithelial cells, neuronal cells, and muscle-type of cells.’32

Transient coexpression of cytokeratin and vimentin in differentiating rat sertoli cells

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.

Expression of cytokeratin polypeptides in mouse oocytes and preimplantation embryos.

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.

Keratin in the epithelial-like cells of classical biphasic synovial sarcoma

SummaryFour 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 differentation in the glandular elements of biphasic synovial sarcomas, and are consistent with earlier ultrastructural observations suggesting epithelial properties of these cells.

Parietal and visceral endoderm differ in their expression of intermediate filaments.

Two layers of extra‐embryonic endoderm, viz. the parietal endoderm (PE) and the visceral endoderm (VE), arise in the mouse embryo shortly after implantation. Both cell populations apparently originate from the primitive endoderm of the blastocyst. While the endoderm differentiation has been studied both in the embryo and in the embryonal carcinoma model system, the investigation has been hampered by the paucity of unequivocal markers of differentiation, especially in the case of the PE. Here we show that the PE and VE of mouse conceptuses differ in their expression of intermediate filaments: while both cell types contain cytokeratin, expression of vimentin was only revealed in the cells of the PE. The association between the differentiation of PE and the appearance of vimentin filaments is discussed.

Fluorochrome-coupled lectins reveal distinct cellular domains in human epidermis.

The distribution of saccharide moieties in human interfollicular epidermis was studied with fluorochrome-coupled lectins. In frozen sections Concanavalin A (Con A), Lens culinaris agglutinin (LCA), Ricinus communis agglutinin I (RCAI), and wheat germ agglutinin (WGA) stained intensively both dermis and viable epidermal cell layers, whereas peanut agglutinin (PNA) bound only to living epidermal cell layers. Ulex europaeus agglutinin I (UEAI) bound to dermal endothelial cells and upper cell layers of the epidermis but left the basal cell layer unstained. Dolichos biflorus agglutinin (DBA) bound only to basal epidermal cells, whereas both soybean agglutinin (SBA) and Helix pomatia agglutinin (HPA) showed strong binding to the spinous and granular cell layers. On routinely processed paraffin sections, a distinctly different staining pattern was seen with many lectins, and to reveal the binding of some lectins a pretreatment with protease was required. All keratin-positive cells in human epidermal cell suspensions, obtained with the suction blister method, bound PNA, whereas only a fraction of the keratinocytes bound either DBA or UEAI. Such a difference in lectin binding pattern was also seen in epidermal cell cultures both immediately after attachment and in organized cell colonies. This suggests that in addition to basal cells, more differentiated epidermal cells from the spinous cell layer are also able to adhere and spread in culture conditions. Gel electrophoretic analysis of the lectin-binding glycoproteins in detergent extracts of metabolically labeled primary keratinocyte cultures revealed that the lectins recognized both distinct and shared glycoproteins. A much different lectin binding pattern was seen in embryonic human skin: fetal epidermis did not show any binding of DBA, whereas UEAI showed diffuse binding to all cell layers but gave a bright staining of dermal endothelial cells. This was in contrast to staining results obtained with a monoclonal cytokeratin antibody, which showed the presence of a distinct basal cell layer in fetal epidermis also. The results indicate that expression of saccharide moieties in human epidermal keratinocytes is related to the stage of cellular differentiation, different cell layers expressing different terminal saccharide moieties. The results also suggest that the emergence of a mature cell surface glycoconjugate pattern in human epidermis is preceded by the acquisition of cell layer-specific, differential keratin expression.

Activation of complement by cytoskeletal intermediate filaments.

INTEREST in cytoplasmic intermediate (10 nm) filaments has grown since the successful immunohistochemical differentiation of these filaments from microtubules and microfilaments1,2. Despite the widespread occurrence of intermediate filaments in various cell types, little is known about their function1,3,4. A cytoskeletal nucleus-anchoring role has been suggested based on their intracellular distribution, their connections to the Plasma membrane and nucleus, and their marked insolubility in detergents and salt solutions5,6. The presence of autoantibodies against intermediate filaments in the sera of patients7,8 suggests that these filaments can be involved in pathogenetic processes. We report here that in studies on possible pathogenetic mechanisms involving intermediate filaments, antibody-independent binding of complement components to intermediate filaments has been observed. Initial observations were made using cultured human embryonal fibroblasts, as cytoplasmic filaments present in these cells have been characterised by using both immunological markers and drugs capable of selectively altering the morphology of microfilaments, intermediate filaments and microtubules1,7. Subsequent studies showed complement binding to similar cytoplasmic structures of other cultured cells and also cells in tissue sections.

Histogenesis of Ewing’s sarcoma

SummaryFour cases of Ewing’s sarcoma, three in bone and one from an extraskeletal site, were studied immunohistologically using monospecific antibodies against intermediate filament proteins of keratin, vimentin, desmin and neurofilament types. All cases were also evaluated for the presence of Factor VIII-related antigen (FVIIIR:Ag) and for the binding of Ulex europaeus I lectin (UEA I), both of which are endothelial markers. In all cases the tumor cells contained vimentin but not keratin, desmin or neurofilaments. The tumor cells could not be decorated with either anti-FVIIIR:Ag or UEA I, whereas the vascular endothelium was positive for both markers. The vimentin-positivity indicates a mesenchymal derivation of Ewing’s sarcoma, while the lack of endothelial markers argues against the proposed endothelial origin of this tumor.

VHS domain -- a longshoreman of vesicle lines.

The VHS ( ps‐27, rs and TAM) domain is a 140 residue long domain present in the very NH2‐terminus of at least 60 proteins. Based on their functional characteristics and on recent data on the involvement of VHS in cargo recognition in trans‐Golgi, VHS domains are considered to have a general membrane targeting/cargo recognition role in vesicular trafficking. Structurally, VHS is a right‐handed superhelix of eight helices with charged surface patches probably serving as sites of protein–protein recognition and docking.

Glomus tumor cells: evaluation of smooth muscle and endothelial cell properties.

SummarySix cases of glomus tumor in superficial soft tissues were investigated immunohistochemically for the presence of different types of intermediate filaments, myosin, laminin, a basal lamina glycoprotein, and the endothelial cell markers, factor VHI-related antigen (FVIIIR:Ag) and Ulex europaeus I lectin (UEA I) binding sites. The tumor cells appeared to contain only vimentin, the fibroblast-type of intermediate filament protein. They were also positive for myosin, and were invested by laminin-positive basal lamina-like material, but did not express endothelial cell markers. Ultrastructural studies revealed prominent arrays of both intermediate filaments and microfilaments, the latter resembling the myofilament bundles seen in smooth muscle cells. The results show that glomus tumor cells resemble smooth muscle cells in their content of myosin and in some ultrastructural features. In their lack of desmin, however, they differ from most types of smooth muscle cell, although they are similar in this respect to some vascular smooth muscle cells.