Granular cell tumors: evidence for heterogeneous tumor cell differentiation

Eighteen granular cell tumors from various sites were examined with antisera directed against protein S-100, neuron specific enolase (NSE), alpha-1-antichymotrypsin, and alpha-1-antitrypsin, glial fibrillary acidic protein (GFAP), lysozyme, factor VIII-related antigen, myoglobin and vimentin, as well as with a monoclonal antibody (lu-5) directed against a panepithelial marker. The immunocytochemical reaction pattern of the tumors was heterogeneous. The brain and pituitary tumors and one thyroid tumor reacted for alpha-1-antichymotrypsin and alpha-1-antitrypsin, but not for S-100 protein and NSE. However, tumors from other sites showed immunoreactions for S-100 protein and NSE and some also for vimentin. Reactions for alpha-1-antichymotrypsin and alpha-1-antitrypsin were not observed. All other reactions were similarly negative. We conclude that the morphologically homogeneous group of granular cell tumors is biologically heterogeneous.     

Immunohistochemical study of sensory nerve formations in human glabrous skin.

The sensory nerve formations (or corpuscles) of normal human glabrous skin from hand and fingers, obtained by punch biopsies, were studied by the streptavidin-biotin method using monoclonal antibodies directed against neurofilament protein (NFP), S-100 protein, glial fibrillary acidic protein (GFAP), cytokeratins, and vimentin. NFP immunoreactivity (IR) was observed in the central axons of most sensory formations, while S-100 protein IR was restricted to non-neuronal cells forming the so-called inner cells core or lamellar cells. Furthermore, vimentin IR was found in the same cells of Meissner's and glomerular corpuscles. None of the sensory nerve formations were stained for GFAP or keratin. The present results suggest that the main nature of the intermediate filaments of the non-neuronal cells of sensory nerve formations from human glabrous skin is represented by vimentin and not by GFAP. Thus, our findings suggest that lamellar and inner core cells of SNF are modified and specialized Schwann cells and not epithelial or perineurial derived cells.     

Immunohistochemical study of granular cell tumors of the neurohypophysis.

Six granular cell tumors (GCT) of the neurohypophysis were studied by immunohistochemical techniques. They were all labeled by peanut lectin (Arachis hypogaea) and three showed reactivity for S-100 protein. Unlike extracranial GCT, neuron specific enolase (NSE), myelin basic protein (MBP) and vimentin were not detected in the tumor cells. Glial fibrillary acidic protein (GFAP), keratin and desmin were also not observed. On the other hand, some showed reactivity for alpha-1-antitrypsin (AAT), alpha-1-antichymotrypsin (AAC) and cathepsin B. These results suggest that neurohypophysial GCT have some features different from extracranial GCT and that they may not be derived from Schwann cells.     

Expression of immunoreactivity for Ca-binding protein, spot 35 in the interstitial cell of the rat pineal organ

Summary In the rat pineal organ numerous stellate cells exhibited intense immunoreactivity for calcium-binding spot 35 protein. Because of their peculiar shape and ultrastructure, identical to those of intrapineal S-100-immunoreactive cells, the spot 35-immunoreactive stellate cells were identified as the interstitial cells. The comparison of the morphology and population density of spot 35-, S-100-, and GFAP (glial fibrillar acidic protein)-immunoreactive cells, suggests that spot 35-immunoreactive cells represent a major subpopulation of the interstitial cells, all of which are S-100-immunoreactive and generally considered to be of glial nature, while GFAP-immunoreactive cells represent a minor subpopulation of the interstitial cells located in the proximal part close to the pineal stalk. This is the first report describing the occurrence of the calcium-binding protein in cells of glial nature.To whom all correspondence should be sent.     

Different distribution of S-100 protein and glial fibrillary acidic protein (GFAP) immunoreactive cells and their relations with nitrergic neurons in the human fetal small intestine.

The appearance, distribution and some histochemical features of non-neuronal cells (NN cells) associated with the myenteric plexus of human fetal small intestine have been studied by means of S-100 protein and GFAP immunocytochemistry between the 10th and 17th week of gestation. In addition, double labelling immunocytochemistry using an antibody raised against a constitutive isoform of nitric oxide synthase (bNOS) in combination with an S-100 protein antibody was applied to investigate the morphological relations between NN cells and nitrergic neurons in the developing gut wall. Cells with immunoreactivity for both glial-specific proteins are already present in the 10th week of gestation. While cells with S-100 protein immunoreactivity are located within the circular muscle layer as well as in the myenteric, and submucous plexuses, cells with GFAP immunopositivity are mainly restricted to the side of the myenteric plexus adjacent to the longitudinal muscle layer. In contrast to the dense network formed by S-100 protein immunopositive structures the GFAP immunopositive cells appear singly and do not connect into a network. Double-labelling immunocytochemistry reveals nitrergic fibers (NOS-IR) in close relation to the S-100 protein immunoreactive glial network. NOS-IR varicosities are in close association with the surface of those cells both in the circular muscle layer (CM) and in the tertiary plexus. It is concluded that two populations of NN cells with different locations and different immunohistochemical characters appear and develop together with the enteric ganglia in the human fetal intestine. The close morphological relation of NOS-IR fibers with S-100 protein immunopositive cellular network indicate a functional relationship between S-100 protein immunopositive cells and the nitrergic nerves during the early development of human enteric nervous system (ENS).     

Immunohistochemical study of granular cell tumors of the neurohypophysis

Six granular cell tumors (GCT) of the neurohypophysis were studied by immunohistochemical techniques. They were all labeled by peanut lectin (Arachis hypogaea) and three showed reactivity for S-100 protein. Unlike extracranial GCT, neuron specific enolase (NSE), myelin basic protein (MBP) and vimentin were not detected in the tumor cells. Glial fibrillary acidic protein (GFAP), keratin and desmin were also not observed. On the other hand, some showed reactivity for alpha-1 -antitrypsin (AAT), alpha-1-antichymotrypsin (AAC) and cathepsin B. These results suggest that neurohypophysial GCT have some features different from extracranial GCT and that they may not be derived from Schwann cells.     

Antibodies to neurofilament protein and other brain proteins reveal the innervation of peripheral organs

Summary Monoclonal and polyclonal antibodies to neurofilament proteins, neuron-specific enolase, glial fibrillary acidic protein and S-100 have been used to demonstrate nerves, ganglion cells and the supportive glial system of the innervation of various organs. The female genitalia, the urinary tract, the respiratory system, the pancreas, the heart and the skin of several mammalian species, including rat, mouse, guinea pig, cat, pig, monkey and man were fixed in parabenzoquinone and portions of each organ were snap frozen. Serial or free-floating thick cryostat sections were stained using indirect immunofluorescence and peroxidase anti-peroxidase immunocytochemistry. In addition, the newly described and highly sensitive immunogold-silver staining technique was used on Bouin's-fixed and wax-embedded tissues.Antibodies to neurofilament proteins seemed to react with neuronal structures in all the species studied. Alternately stained serial sections showed a similar distribution of neurofilament proteins and neuron-specific enolase-containing nerves. Neuron-specific enolase staining had a diffuse appearance and was found to be highly variable, indicating that the neuron-specific enolase content might be related to the physiological state of the nerves and ganglion cells, whereas antibodies to neurofilament protein gave a consistently intense and very clear picture of the ganglion cells and nerve fibres. Antibodies to S-100 stained supportive elements of the peripheral nervous system in all tissues examined, whereas antibodies to glial fibrillary acidic protein were more selective.Abbreviations GFAP glial fibrillary acidic protein - NSE neuron-specific enolase - PBS phosphate-buffered saline - PAP peroxidase anti-peroxidase - FITC fluorescein-isothiocyanate     

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.     

Immunohistochemical co-localization of glycogen phosphorylase with the astroglial markers glial fibrillary acidic protein and S-100 protein in rat brain sections.

Immunofluorescence double-labelling and immunoenzyme double-staining methods were used to examine the location of glycogen phosphorylase brain isozyme with the astrocyte markers glial fibrillary acidic protein (GFAP) and S-100 protein in formaldehyde-fixed, paraffin-embedded slices from adult rat brain. Astrocytes in the cerebellum and the hippocampus, which express GFAP or S-100 protein immunoreactivity, show glycogen phosphorylase immunoreactivity. Regional intensity and intracellular distribution of the three antigens vary characteristically. In ependymal cells, glycogen phosphorylase immunoreactivity is co-localized with S-100 protein immunoreactivity, but not with GFAP immunoreactivity. These findings confirm that glycogen phosphorylase in the rat brain is exclusively localized in astrocytes and ependymal cells. All astrocytes, as far as they express GFAP or S-100 protein, do contain glycogen phosphorylase.     

Immunohistochemical co-localization of glycogen phosphorylase with the astroglial markers glial fibrillary acidic protein and S-100 protein in rat brain sections

Summary Immunofluorescence double-labelling and immunoenzyme double-staining methods were used to examine the location of glycogen phosphorylase brain isozyme with the astrocyte markers glial fibrillary acidic protein (GFAP) and S-100 protein in formaldehydefixed, paraffin-embedded slices from adult rat brain. Astrocytes in the cerebellum and the hippocampus, which express GFAP or S-100 protein immunoreactivity, show glycogen phosphorylase immunoreactivity. Regional intensity and intracellular distribution of the three antigens vary characteristically. In ependymal cells, glycogen phosphorylase immunoreactivity is co-localized with S-100 protein immunoreactivity, but not with GFAP immunoreactivity. These findings confirm that glycogen phosphorylase in the rat brain is exclusively localized in astrocytes and ependymal cells. All astrocytes, as far as they express GFAP or S-100 protein, do contain glycogen phosphorylase.     

Differential localization of "brain-specific" S-100 and its subunits in rat salivary glands

In the rat, the S-100 antigens in the submandibular gland were found to be immunochemically identical with those in the brain (glial cells) when compared using crossed immunoelectrophoresis. Specific antibodies against the S-100a non-beta and against the S-100 beta subunit were prepared from antibodies against crude S-100 protein and from S-100 components (S-100a and b) by affinity chromatography. In the rat salivary glands a differential distribution of subunit immunoreactivity was clearly evidenced using indirect immunofluorescence. Certain intercalated duct cells of the submandibular gland as well as Schwann cells contained the S-100 beta subunit immunoreactivity exclusively, while other duct cells in parotid, submandibular, and sublingual glands contained S-100a non-beta subunit immunoreactivity. Both subunits were present in astrocytes and ependymal cells. The immunocytochemical localization of alpha and beta subunits is a promising technique for the classification of various types of S-100-containing cells.     

Immunohistochemical localization by monoclonal antibodies of S-100 alpha and beta proteins in mixed tumours and adenomas of the skin

A study using monoclonal antibodies was made to evaluate the immunohistochemical localization of S-100 protein subunits alpha and beta in a total of 41 mixed tumours and adenomas of sweat gland origin. Normal eccrine glands showed positive staining for S-100 alpha in the secretory portion and in epithelial cells located in the transitional area from the coiled duct to the intraepidermal duct, as well as granular deposition of S-100 beta at the luminal surface of the secretory coil and duct. The myoepithelial cells were negative for S-100 alpha and beta. In mixed tumours, the tumour cells were round or oval in shape and displayed markedly positive staining for S-100 alpha and slightly positive or negative staining for S-100 beta. S-100 alpha staining in clear cell tumours was typically more intense than in any other sweat gland tumour. It is possible that clear cell tumours may arise from the transitional area of sweat glands. Spindle cell tumours displayed on abundance of S-100 alpha subunits but little S-100 beta. Occasional spindle cells located in the outer layer of tubular structures within tumours gave positive S-100 alpha staining. This result was different from that seen in pleomorphic salivary adenomas. Cells having undergone chondroidal changes revealed a positive S-100 reaction.     

Morphological and neurochemical diversity of neuronal nitric oxide synthase-positive amacrine cells in the turtle retina

By gently scraping off the surface of the lateral ventricles of adult bovine brains, we obtained sheets containing the ependymal layer and some attached sub-ependymal cells. Explants were cultured in serum-free medium or in two media enriched with 20% fetal calf serum or 20% adult bovine cerebrospinal fluid, and processed for different time intervals from 4 h to 60 days. For characterization of the ependymal cells we used antisera against S-100 protein, vimentin and glial fibrillary acidic protein (GFAP). For comparison, the ependyma of adult bovines and of fetuses from days 60 to 120 post coitum was studied in situ. The adult ependyma consisted of a ciliated, cuboid cell monolayer with short basal processes; it displayed S-100 immunoreactivity but only scarce deposits of vimentin and no GFAP. The fetal ependyma had the appearance of a pseudostratified epithelium with elongated nuclei and basal processes containing S-100 and vimentin from day 80 post coitum and GFAP from day 100 post coitum. In explants, no differences were seen between the three culture media; the ependyma became pseudostratified, developed basal processes and showed increasing amounts of S-100 and vimentin first, and subsequently also GFAP. These changes were concomitant with the onset of mitotic activity in the subependymal layer leading to the production of numerous cells. The morphological and immunocytochemical features of ependymal cells in cultured explants resembled those of fetal ependyma. Our results indicate that the culture of ependymal explants from adult bovine lateral ventricles is an useful model system for morphological and functional studies of the ependyma and for the analysis of cell proliferation in the subependymal layer.     

Effect of sodium butyrate on S-100 protein levels and the cAMP response

Sodium butyrate (NaB), when added to cell cultures, produces a variety of morphological and biochemical changes. We examined its effects, in nM concentrations, on the expression of two glioma cell-associated proteins, glial fibrillary acidic protein (GFAP) and S-100 protein in human glioma-derived cell line (RF), and of S-100 protein in the C6 rat glioma cell line. GFAP levels decreased by about 50% in the RF cell line, and S-100 protein levels decreased protein levels decreased by about 40% after treatment with 1 mM NaB for 48 h. In the C6 rat glioma cell line, isoproterenol with theophylline was found to increase S-100 levels by two-fold over basal levels. NaB was found to inhibit the induction of S-100 protein but exhibited no effect on the basal levels of the protein. Other short chain fatty acids, including sodium propionate and sodium isobutyrate, exhibited partial inhibitory activity. NaB, at an EC50 of 1 mM, was also found to inhibit both the beta-adrenergic and the forskolin-mediated increase in cAMP levels in these cells. This suggests that NaB may inhibit cells from expressing S-100 protein by attenuating cAMP levels.     

Antigenic profile of the human lacrimal gland.

The antigenic profile of 13 normal formalin-fixed, paraffin-embedded human main and accessory lacrimal glands, biopsied from patients aged 11 to 78 years, was studied using a panel of 27 polyclonal and monoclonal antibodies. Secretory cells of lacrimal acini reacted with antibodies to S-100 protein and simple epithelium-type cytokeratins CK 7, CK 8, CK 18, and CK 19. Their luminal membranes were labeled with antibodies to carcinoembryonic antigen, epithelial membrane antigen, and epithelial glycoproteins recognized by Ber-EP4. Myoepithelial cells were often immunopositive for S-100 protein, vimentin, glial fibrillary acidic protein (GFAP), and alpha-smooth muscle actin. More rarely, they reacted with antibodies recognizing CK 5, CK 13, and CK 14, which consistently labeled the basal cells of lacrimal ducts. Unlike myoepithelial cells, basal ductal cells were immunopositive for CK 7, CK 8, CK 18, and CK 19. In main excretory ducts, dendritic melanocyte-like cells co-expressing vimentin and S-100 protein intermingled with ductal epithelial cells. The luminal cells of lacrimal ducts basically paralleled secretory cells in their antigenic profile, although they lacked Ber-EP4 and were immunopositive for CK 4. Antibodies to neuron-specific enolase and synaptophysin reacted with nerve fibers among negatively reacting secretory acini. This antigenic profile closely parallels that of salivary glands and provides a basis for studies of lacrimal gland pathology.     

Immunohistochemical demonstration of neuronal and astrocytic markers and oncofoetal antigens in retinoblastomas.

General opinion is that retinoblastomas, though not everyone agrees with that view. Some authors suggest that retinoblastomas are derived from a primitive retinal cell able to differentiate into both neuronal and glial cell lines. The aim of the present work was to study immunohistochemically the expression of neuronal and astrocytic markers in retinoblastomas and at the same time the presence of the oncofoetal antigens carcinoembryonic antigen (CEA) and alpha Foeto Protein (AFP), since patients with retinoblastomas often show high oncofoetal antigen in serum levels. For this purpose we employed the streptavidin-biotin immunoperoxidase technique in 13 cases of retinoblastoma to evaluate the presence and distribution of neuron-specific enolase (NSE), neurofilament protein (NF), glial fibrillary acidic protein (GFAP), S-100 protein, CEA and AFP. All 13 tumours studied stained for NSE. Seven of them showed GFAP- and S-100 positive perivascular glial cells as well as cells distributed randomly in the tumour that were interpreted as non tumour cells. All 13 retinoblastomas lacked detectable NF, CEA, and AFP. These results support the idea that retinoblastomas are neuronal tumours, although retinal glial cells may become incorporated in the tumour and proliferate in response to the tumour.     

Acquisition of blood--tissue barrier--supporting features by hepatic stellate cells and astrocytes of myofibroblastic phenotype. Inverse dynamics of metallothionein and glial fibrillary acidic protein expression

A number of similarities between astrocytes and hepatic stellate cells (HSC) rose the question whether or not the protective barrier features of blood-tissue interface may be provided by HSC as well. To test this hypothesis, we investigated the presence of metallothionein (MT), a functional marker of blood--brain barrier, in HSC in situ and in cell culture and compared the results with those obtained with astrocytes. The dynamics of MT expression in cultured astrocytes and HSC was investigated by simultaneous labelling of the cells with a monoclonal antibody (MAb MT) against a lysine-containing epitope of the cadmium-induced monomer of MT-I from rat liver and antiserum against glial fibrillary acidic protein (GFAP). Cell activation was estimated by the presence of smooth muscle alpha-actin (SMAA). In immunoblotting, MAb MT recognized monomeric MT protein and proteins in the 30-kDa range; both bands were pronounced in brain and barely visible in liver homogenates. In situ, MAb MT reacted with very few perivascular cells situated in the parenchyma of the liver. Double immunolabelling of brain slices with MAb MT and antiserum against GFAP showed large areas of brain containing cells expressing both MT and GFAP. However, there were also regions in the brain where the cells produced solely GFAP or MT. In liver cell culture, MT was absent from HSC and hepatocytes in early periods of cultivation, during which the cells maintained their original features; however, MT was expressed strongly in HSC during their activation under prolonged culture conditions. Inversely, in astrocytes MT was expressed during early culturing and disappeared from the cells together with SMAA in late culture when GFAP was upregulated. These results suggest that the acquisition of myofibroblastic features by perivascular cells empowers them to establish a protective blood-tissue permeability barrier. In addition, this study shows that, at least in cell culture, an enrichment of perivascular cells in GFAP results in the disappearance of protective functions.     

Quantitative immunochemistry on neuronal loss, reactive gliosis and BBB damage in cortex/striatum and hippocampus/amygdala after systemic kainic acid administration

Cell specific markers were quantified in the hippocampus, the amygdala/pyriform cortex, the frontal cerebral cortex and the striatum of the rat brain after systemic administration of kainic acid. Neuron specific enolase (NSE) reflects loss of neurons, glial fibrillary acidic protein (GFAP) reflects reactive gliosis, and brain levels of serum proteins measures blood-brain-barrier permeability. While the concentration of NSE remained unaffected in the frontal cerebral cortex and the striatum, their GFAP content increased during the first three days. In the hippocampus and amygdala, NSE levels decreased significantly. GFAP levels in the hippocampus were unaffected after one day and decreased in the amygdala/pyriform cortex. After that, GFAP increased strikingly until day 9 or, in the case of amygdala/pyriform cortex, even longer. This biphasic time course for GFAP was accompanied by a decrease of S-100 during days 1-9 followed by a significant increase at day 27 above the initial level. The regional differences in GFAP and S-100 could result from the degree of neuronal degeneration, the astrocytic receptor set-up and/or effects on the blood-brain barrier.     

Significance of S-100 protein immunostaining in the immunohistological analysis of normal and neoplastic lymphoid tissues--an appraisal

S-100 protein is a heterogeneous fraction of dimeric polypeptides (alpha and beta subunits) that can exist in different combination forms within the various tissues. Concerning the S-100 protein immunodetection within lymphoid tissue, the heterogeneity of the S-100 antigen, the tissue quality (frozen or paraffin-embedded after treatment with different fixatives) and the treatment of the tissue with different immunostaining methods and antibodies of different nature, all make for inconsistent results obtained in the immunohistological studies reported in the literature. Most of the S-100-positive cells of the lymphoreticular system are dendritic cells involved in the immune response (interdigitating reticulum cells, Langerhans cells, and follicular dendritic reticulum cells), other S-100-positive cells belonging to the mononuclear/phagocytic system. S-100 protein immunostaining may be used as a helpful immunohistological diagnostic clue to certain malignancies of the immune system (follicular center cell lymphomas) on the basis of their specifically related dendritic cell microenvironment. In addition to monoclonal antibodies for the immunophenotypic characterization of dendritic cells and macrophages and to enzyme reactions, the combined use of anti-S-100 antibodies specific for each of the S-100 protein subunits, tested with sensitive procedures, would be a very useful tool in the attempt to classify the proliferative disorders of dendritic cells and macrophages.     

Immediate early gene IEX-1 induces astrocytic differentiation of U87-MG human glioma cells

The immediate early response gene IEX-1 is involved in the regulation of apoptosis and cell growth. In order to increase the apoptotic sensitivity to chemotherapeutic drugs and gamma-ray, we attempted to establish U87-MG human glioma cell line expressing IEX-1. Unexpectedly, however, transfection of IEX-1 into U87-MG glioma cells resulted in morphological changes to astrocytic phenotype and increase in glial differentiation marker proteins, S-100 and glial fibrillary acidic protein (GFAP). Glial cell differentiation was used to examine in rat C6 glioma cell line, since this cell line express astrocytic phenotypes by increase in intracellular cAMP concentration. Stimulation of human U87-MG glioma cells by membrane-permeable dibutyryl cAMP (dbcAMP) not only elicited their morphological changes but also induced expression of IEX-1 as well as S-100 and GFAP. H89, an inhibitor of protein kinase A (PKA), blocked dbcAMP-induced morphological changes of U87-MG cells and expression of IEX-1. In contrast, morphological changes and expression of S-100 and GFAP induced by IEX-1 were not affected by H89. Morphological changes induced by dbcAMP were totally abolished by functional disruption of IEX-1 expression by anti-sense RNA. These results indicate that IEX-1 plays an important role in astrocytic differentiation of human glioma cells and that IEX-1 functions at downstream of PKA.