Expression and distribution of vimentin and keratin filaments in heterokaryons of human fibroblasts and amnion epithelial cells

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.     

Regulation of vimentin expression in cultured human mammary epithelial cells

Using five different monoclonal antibodies to vimentin, we have examined the expression of vimentin in cryostat sections and serum-free cultures of normal human breast tissue. In cryostat sections, myoepithelial cells as well as stromal cells showed immunoreactivity to vimentin, irrespective of the antibody used. In contrast, luminal epithelial cells were negative for vimentin, but positive for keratin K18. In culture, myoepithelial cells showed immunoreactivity to vimentin from their first appearance in monolayer. Moreover, a fraction of luminal epithelial cells expressed vimentin in addition to keratin K18. We found a clear, reversible correlation between proliferation, determined by incorporation of [3H]-TdR, and induction of vimentin in the luminal epithelial cells. Thus, in growth-stimulated cultures on a medium containing cholera toxin (CT), epidermal growth factor (EGF), transferrin (Tf), hydrocortisone (H) and insulin (I), the fraction of vimentin-positive luminal epithelial cells increased, while it decreased within 14 days from approximately 36% to 3% on a medium containing CT and EGF, only. We therefore conclude: (1) vimentin is constantly expressed in myoepithelial cells in situ and in vitro, and (2) expression of vimentin in luminal epithelial cells in vitro is not a result of monolayer cultivation as such, but rather associated with the increased growth rate seen in culture.     

Intermediate filament expression in non-neoplastic pituitary cells.

Fifty-one non-neoplastic human pituitary glands, including examples with Crooke's hyalinization or amyloidosis, were examined by an immunoperoxidase method using antibodies to keratin, vimentin, neurofilaments (NFs), glial fibrillary acidic protein (GFAP), desmin, actin, S-100 protein and a variety of pituitary hormones. It was confirmed that most of the epithelial cells in the pituitary gland express keratin immunoreactivity. These cells included endocrine cells in the anterior lobe, endocrine cells and squamous metaplastic cells in the pars tuberalis, columnar and ciliated epithelia forming follicular structures and salivary-type epithelium in the pars intermedia, and anterior lobe cells infiltrating the posterior lobe. This study also demonstrated that keratin and NFs may be co-expressed in endocrine cells in the pituitary anterior lobe, that keratin, vimentin and GFAP may be co-expressed in the epithelial cells forming cyst-like follicle in the pars intermedia, and that vimentin and GFAP may be co-expressed in folliculo-stellate cells and pituicytes. In addition, the GFAP and S-100 protein-negative high columnar epithelium in the pars intermedia tended to be positive for adrenocorticotropic hormone and melanocyte stimulating hormone, while the low columnar epithelium with the co-expression of GFAP and S-100 protein was negative for pituitary hormones.     

Early expression of vimenti in human mammary cultures

Summary The intermediate filaments of most epithelial cells in vivo consist solely of cytokeratins. Using monoclonal antibodies to vimentin or keratin, we have examined the expression of vimentin in homologous specimens of frozen tissue sections and primary cultures of normal human mammary epithelium. In frozen sections, only epithelial cells reacted with the antikeratin antibody, whereas antivimentin reactivity was associated with stromal cells. All epithelial cultures were positive for cytokeratin and in addition coexpressed vimentin as strongly as cultured fibroblasts and as early as the 4th d after initiation of the culture. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis of cytoskeletal preparations of secondary cultures of normal mammary epithelium have also demonstrated the appearance of a moiety identical to the vimentin found in cultured fibroblasts. Our observations are consistent with the hypothesis that vimentin expression is induced, possibly as a result of changes in cell shape or growth rate, when cells are freed from three-dimensional restirctions imposed by the tissue of origin     

Immunophenotypic properties and estrogen dependency of budding cell structures in the developing mouse mammary gland

Abstract. The initial phase of growth of the parenchymal component of the mouse mammary gland is ductal clongation, which is mainly accomplished by proliferating cells in a specialized structure termed end bud. End buds are composed of multiple layers of epithelial cells (so called body cells) which are capped by a single layer of morphologically unique cells termed cap cells.
We sought to examine the interrelationship between cap cells and other epithelial cell subclasses using a variety of antibodies to different keratin proteins and also antibodies to vimentin, actin and collagen IV. An extensive immunohistochemical characterization of the epithelial components of the developing and differentiating mammary gland demonstrated that cap cells were devoid of any immunohistochemically - detectable keratins but were positive for collagen IV. In contrast, the majority of cells in the end bud along with the luminal epithelial and myoepithelial cells were keratin positive. The body cells of the end bud were the only cells which were positive for antibody to keratin 6, a keratin which previously has been reported to be expressed in proliferating mammary epithelial cells. In addition, estrogen receptor was localized only to epithelial cells of ducts, alvcoli and body cells of end buds, but not to cap cells or myoepithelial cells. We interpret these results to suggest that cap cells are not totpotent stem cells but rather cells specialized in paving the way for ductal elongation as well as serving as precursors to myoepithelial cells.     

Integration of different keratins into the same filament system after microinjection of mRNA for epidermal keratins into kidney epithelial cells

We have isolated poly (A)+ RNA, highly enriched in keratin mRNA from bovine muzzle epidermis, and injected it into epithelial cells of a different type, i.e., cultured kidney epithelial cells of the same (MDBK) or taxonomically distant (PtK2) species. Both recipient cell lines contain keratin polypeptides that are different from those present in epidermal cells. Using keratin subtype-specific antibodies in immunofluorescence and immunoelectron microscopy, we show that foreign keratin mRNAs when injected into a different type of epithelial cell can recruit polyribosomes and are translated together with the keratin mRNAs of the host cell. Foreign epidermal keratins are excluded from vimentin filaments and other structures but readily coassemble with the endogenous keratins and appear to be integrated into the meshwork of the preexisting kidney-type keratin filaments. Our observations indicate that different sets of keratin polypeptides from the same or different species can coassemble in the living cell into a common filament system. Thus we have developed a procedure that allows experimental alteration of the intermediate filament cytoskeleton within living epithelial cells.     

奶山羊乳腺上皮细胞的分离、培养及鉴定

应用组织块培养法高密度培养、连续传代法建立西农萨能奶山羊乳腺上皮细胞体外培养体系,通过生长曲线绘制、核型分析、免疫荧光染色 (角蛋白、上皮膜抗原、波形蛋白、β-酪蛋白)、油红染色及β-酪蛋白基因的RT-PCR分析进行培养细胞鉴定。实验结果表明细胞生长曲线为典型的S型,染色体数目众数为60,细胞角蛋白、上皮膜抗原、波形蛋白、β-酪蛋白表达均呈阳性,油红染色后可见细胞质内的脂滴,且细胞表达酪蛋白mRNA。说明运用本方法培养的细胞为正常的乳腺上皮细胞,并具有一定的泌乳功能。     

Vimentin and keratin intermediate filament systems in cultured PtK2 epithelial cells are interrelated.

Certain cultured epithelial cells contain separate vimentin and keratin-type intermediate filament networks. The intracellular injection of monoclonal antibodies directed against either vimentin or keratin filaments into PtK2 cultured epithelial cells specifically disrupted the organization of both filament types. Neither antibody had any effect when injected into cells which, while containing vimentin or keratin filaments, lacked the specific filament type which that antibody recognized. These experiments suggest that keratin and vimentin filament networks are associated in some way with one another.     

Various keratin antibodies produce immunohistochemical staining of human myocardium and myometrium

Various polyclonal and monoclonal antibodies to keratins were used to stain different human muscle tissues by paired immunofluorescence and the unlabelled antibody peroxidase-anti-peroxidase method. In the myocardium, distinct coloration of the intercalated discs was produced by two polyclonal reagents to human epidermal keratins but not by two monoclonal antibodies to cytokeratins from pig renal tubular cells. In the myometrium--mainly in the middle layer of the uterine wall--cytoplasmic coloration of a varying fraction of the smooth muscle bundles was produced, especially by one of the polyclonal and by both monoclonal reagents. The staining was often confined to the perinuclear region. The keratin-positive myometrial cells usually coexpressed vimentin and actin in various proportions. These findings indicated that intermediate filaments of the keratin type, or antigenically similar elements, are not restricted to cells of epithelial origin. Other types of muscle cells did not react with keratin antibodies, but keratin-positive macrophages were occasionally found in tongue musculature and in inflamed epicardium. Altogether, our observations emphasize that keratin reactivity cannot be considered specific for epithelial (or mesothelial) cells without reservation.     

Microinjection of monoclonal antibodies specific for one intermediate filament protein in cells containing multiple keratins allow insight into the composition of particular 10 nm filaments

Monoclonal antibodies specific for vimentin (V9), keratin 7 (CK 7) and keratin 18 (CK5) have been microinjected into three human epithelial cell lines: HeLa, MCF-7 and RT-4. The effect of the injection on other keratin polypeptides and vimentin filaments has been observed by double label immunofluorescence and in some instances by immunoelectron microscopy using gold labels of different sizes. Microinjection of V9 into HeLa cells causes the vimentin to collapse into a perinuclear cap leaving the keratin filaments unaffected. Injection of CK5 does not affect the vimentin filaments but disrupts the keratin filaments revealing keratin aggregates similar to those seen in some epithelial cell lines during mitosis. The keratin aggregates obtained after microinjection in HeLa contain the keratins 8 and 18 and probably also other keratins, as no residual keratin filaments are observed with a keratin polyclonal antibody of broad specificity. Aggregates in mitotic HeLa cells contain at least the keratins 7, 8, and 18. In MCF-7 cells keratins 8, 18, and 19 are observed in the aggregates seen 3 h after microinjection which, however, show a different morphology from those seen in HeLa cells. In MCF-7 cells a new keratin filament is built within 6 h after the injection which is composed mainly of keratin 8 and 19. The antibody-complexed keratin 18 remains in spherical aggregates of different size. The results suggest that in HeLa cells vimentin and keratin form independent networks, and that individual 10 nm filaments in epithelial cell lines can contain more than two keratins.     

Analysis of thymic stromal cell subpopulations grown in vitro on extracellular matrix in defined medium. I. Growth conditions and morphology of murine thymic epithelial and mesenchymal cells

We report here the successful selective cultivation of murine thymic mesenchymal reticular cells (MTMC) and murine thymic epithelial cells (MTEC) grown on extracellular matrix in the presence of defined medium. The selective growth of these two cell types was based on 1) conditions of tissue disruption and 2) differential growth requirements. Both cell types were dependent on transferrin, high density lipoproteins, insulin, hydrocortisone, and epidermal growth factor, whereas MTMC was dependent also on selenium and 3,5,3'-triiodothyronine. The elimination of single factors or extracellular matrix resulted in specific and different changes in the growth pattern of each cell subpopulation. Cells of both types exhibited the ultrastructural features of high metabolic activity. The epithelial nature of MTEC cultures was defined by bundles of tonofilaments and desmosomes and by positive staining to keratins and negative to vimentin. In addition MTEC were positively stained with mAb to thymic medullary epithelial cells and by Ulex europeus agglutinin, and were able to form Hassall's corpuscles, suggesting their medullary origin. MTEC were also H-2 and Ia positive. In contrast MTMC were positive for vimentin and periodic acid-Schiff, low positive for H-2, and negative for keratin and Ia. Both cells did not contain nonspecific esterase, nor did they phagocytize latex beads. With the use of all these criteria we classified MTEC as epithelial cells from the medullary compartment of the thymus and MTMC as reticular cells of mesenchymal origin.     

Microinjection of IFA antibody induces intermediate filament aggregates in epithelial cell lines but perinuclear coils in fibroblast-like lines.

The murine monoclonal IFA antibody recognizes a conserved sequence present in almost all intermediate filament (IF) proteins. When IFA antibody was injected into 13 different primary or established cell lines, striking differences were detected between epithelial and fibroblastic cell lines. In epithelial cells keratin IFs were broken down within 4 h into numerous spheroid aggregates scattered throughout the cytoplasm. Keratin aggregates were first detected in the cytoplasmic periphery. In contrast, in fibroblastic cells, injection of IFA antibody led to the formation of perinuclear coils of vimentin. IFA antibody at a concentration of greater than 1 mg/ml had to be injected to initiate these transitions. When HeLa cells, which contain separate networks of vimentin and keratin filaments, were injected with IFA antibody, vimentin did not form perinuclear coils but was instead found together with keratin in aggregates. Electron micrographs of HeLa cells injected with IFA antibody showed that the aggregates have diameters between 0.5 and 2.6 microns and resembled the keratin aggregates observed in certain mitotic epithelial cells. Although the ultrastructural studies support an association of some aggregates with desmosomes, aggregates were, however, also induced by injection of IFA antibody into human keratinocytes in low calcium medium under conditions where desmosomes were not present.     

Identification of cultured cell employing specific substance--with special references of "reserve cells" in uterine cervix

Identification of endocervical "reserve cell", which have been regarded as the origin of squamous cell carcinoma of the uterine cervix, was attempted employing immunohistochemically specific substances. The antigenicity of keratin, squamous cell carcinoma antigen(SCC), epithelial membrane antigen(EMA), tissue polypeptide antigen(TPA), vimentin, secretory component(SC) and placental alkaline phosphatase(PLAP) was investigated in histological preparations as well as cultured cells obtained from primary culture of endocervical tissue. The immunohistochemical findings in histological preparations revealed the following: a strongly positive reaction with TPA, a slightly positive reaction with EMA, a very slightly positive with SCC and PLAP, and a negative reaction with keratin, vimentin and SC. Cultured cells were divided into 4 groups according to their morphological characteristics; among these, small rounded or polygonal cells with a centric single nucleus showed similar immunocytochemical reactions to those of "reserve cells" in histological preparations, indicating that "reserve cell" can be growing in culture. The results obtained suggest that immunohistocytochemical specific substances may be useful to identify cultured cells.     

Various keratin antibodies produce immunohistochemical staining of human myocardium and myometrium

Summary Various polyclonal and monoclonal antibodies to keratins were used to stain different human muscle tissues by paired immunofluorescence and the unlabelled antibody peroxidase-anti-peroxidase method. In the myocardium, distinct coloration of the intercalated discs was produced by two polyclonal reagents to human epidermal keratins but not by two monoclonal antibodies to cytokeratins from pig renal tubular cells. In the myometrium — mainly in the middle layer of the uterine wall — cytoplasmic coloration of a varying fraction of the smooth muscle bundles was produced, especially by one of the polyclonal and by both monoclonal reagents. The staining was often confined to the perinuclear region. The keratin-positive myometrial cells usually coexpressed vimentin and actin in various proportions. These findings indicated that intermediate filaments of the keratin type, or antigenically similar elements, are not restricted to cells of epithelial origin. Other types of muscle cells did not react with keratin antibodies, but keratin-positive macrophages were occasionally found in tongue musculature and in inflamed epicardium. Altogether, our observations emphasize that keratin reactivity cannot be considered specific for epithelial (or mesothelial) cells without reservation.Supported by the Norwegian Cancer Society, Jahre's Fund, and the Norwegian Research Council for Science and the Humanities     

中等纤维在几种人体上皮细胞有丝分裂过程中的行为

By indirect immunofluorescence microscopy and electron microscopy, we studied the behavior of intermediate filaments during mitosis in three human epithelial cell lines, derived from normal epidermis (PcaSE-1, from a cancer patient), stratified epithelium (CNE, from nasopharyngeal carcinoma) and simple epithelium (SPC-A-1 from lung adenocarcinoma) respectively. CNE cells and SPC-A-1 cells express two different intermediate filament systems; keratin filaments and vimentin filaments, but PcaSE-1 cells only express keratin filaments. The keratin filament system in PcaSE-1 cells remained intact and encircled the developing mitotic spindle as the cells entered mitosis. In contrast, in CNE cells and SPC-A-1 cells, keratin filaments appeared to disassemble into amorphous cytoplasmic bodies during mitosis. However, their vimentin filaments remained morphologically intact throughout mitosis. We propose; (1) The disassembly of keratin filaments in mitotic epithelial cells is more or less associated with the degree of their cell malignancy rather than with the abundance of keratin filaments in interphase. (2) Intermediate filaments may be involved in the positioning and/or centering of the spindle during mitosis. (3) The possible function of vimentin filament system in CNE cells is positioning and orientation of chromosomes.     

Two-colour immunofluorescence marker study of pleomorphic adenomas

Extensive use of two-colour immunofluorescence staining for various cell markers in pleomorphic adenoma, revealed three consistent phenotypic features: (1) keratin polypeptide No. 14, which was virtually restricted to myoepithelial cells (MEC) in normal salivary glands, appeared in a large fraction of the tumour cells, suggesting that the principal neoplastic element is derived from MEC or their immediate precursors; (2) a complex co-expression pattern of various cell markers was found, with extensive concurrence of keratin and vimentin in strands of MEC-like and myxoid tumour cells, probably reflecting different degrees of tumour cell differentiation; and (3) two phenotypically distinctive dendritic cell populations were identified, one consisting of keratin positive tumour cells and the other of HLA-DR positive but keratin negative stromal cells. The significance of these findings with regard to the histogenesis and complex morphology of pleomorphic adenoma is discussed.     

中等纤维在几种人体上皮细胞有丝分裂过程中的行为

本文用间接免疫荧光法和电镜术观察了分别来自人表皮(PcaSE-1)、复层上皮(CNE)和单层上皮(SPC-A-1)的3个上皮细胞系的细胞在有丝分裂过程中中等纤维的行为。结果表明,CNE细胞和SPC-A-1细胞表达两种不同类型的中等纤维系统:角蛋白纤维和波形纤维,而PcaSE-1细胞仅表达角蛋白纤维。当细胞进入有丝分裂时,PcaSE-1细胞的角蛋白纤维维持完整的形态且将有丝分裂纺锤体围绕在细胞中央。相反,在CNE细胞和SPC-A-1细胞中,在细胞有丝分裂时,角蛋白纤维解聚成无定形的胞质小体,然而它们的波形纤维始终保持完整的形态。我们认为(1)在分裂上皮细胞中,角蛋白纤维的解聚与细胞的恶性程度有关,而与间期上皮细胞中是否含有丰富的角蛋白纤维无明显关系。(2)在上皮细胞有丝分裂时,中等纤维可能参于纺锤体的定位和趋中。(3)在分裂CNE细胞中,波形纤维的可能功能是染色体的定位和定向。     

Stromal decidualization of endometriosis in the rhesus macaque (Macaca mulatta): a case report

During exploratory laparotomy, a 10-year-old female rhesus macaque was found to have a 6.0 x 9.5 x 2.0-cm multichambered, yellow, cystic mass cranial to the uterus, from which large amounts of opaque, white fluid were discharged into the abdominal cavity. The animal was euthanized, and the body was submitted for gross and histologic evaluation. Sections of the mass examined microscopically consisted of sheets of polygonal to round cells, with well defined cell borders and moderate amounts of eosinophilic cytoplasm. Scattered throughout these cells were few, variably sized glandular structures composed of columnar to cuboidal epithelium. Glandular epithelial cells were positive for keratin, and the sheets of polygonal cells were positive for vimentin and negative for keratin and CD 68. Gross and histologic appearance, immunohistochemical findings, and history of medroxyprogesterone acetate injections were compatible with a diagnosis of stromal decidualization of endometriosis. Subsequent biopsies of similar lesions in other rhesus macaques in the colony being treated with medroxyprogesterone acetate for endometriosis revealed comparable histologic findings.     

Two-colour immunofluorescence marker study of pleomorphic adenomas

Summary Extensive use of two-colour immunofluorescence staining for various cell markers in pleomorphic adenoma, revealed three consistent phenotypic features: (1) keratin polypeptide No. 14, which was virtually restricted to myoepithelial cells (MEC) in normal salivary glands, appeared in a large fraction of the tumour cells, suggesting that the principal neoplastic element is derived from MEC or their immediate precursors; (2) a complex co-expression pattern of various cell markers was found, with extensive concurrence of keratin and vimentin in strands of MEC-like and myxoid tumour cells, probably reflecting different degrees of tumour cell differentiation; and (3) two phenotypically distinctive dendritic cell populations were identified, one consisting of keratin positive tumour cells and the other of HLA-DR positive but keratin negative stromal cells. The significance of these findings with regard to the histogenesis and complex morphology of pleomorphic adenoma is discussed.     

Phenotypic analysis of cultured melanoma cells : Expression of cytokeratin-type intermediate filaments by the M5 human melanoma cell line

Expression of intermediate filament (IF) isotypes was studied in six human and two murine melanoma cell lines. With one exception, these lines expressed IFs only of the vimentin type; neurofilament peptides, desmin and GFAP were not detected. However, the M5 human melanoma line also expressed extensive cytokeratin tonofilament arrays, as visualized by immunofluorescence with a panel of eleven monoclonal antibodies and hetero-antisera to cytokeratins; only the keratin 19-specific antibody BA16 did not react. By 2 D gel electrophoresis, five major keratin peptides were detected (keratins 7, 8, 13, 17 and 18), and an additional 57 kD peptide was detected on immunoblots with several antikeratin antibodies. Also observed in M5 cells was focal collapse of tonofilament arrays in mitotic cells. All the melanoma lines tested were positive for S100; M5 and two other cell lines were also positive for the 220-240 kD neuroectoderm-associated cell-surface differentiation antigen defined by monoclonal antibody UJ 127:11. In all the melanoma cell lines, secretion of extracellular matrix proteins (fibronectin, laminin and collagen type IV) was sparse or absent, and all were negative for the epithelial cell markers HMG-1 and HMG-2. Co-expression of keratin and vimentin by a melanoma cell line is discussed in the light of recent controversy concerning expression of cytokeratins by other neoplasms of putative neuroectodermal origins.