Staining of proteoglycans in mouse lung alveoli. II. Characterization of the Cuprolinic Blue-positive, anionic sites

Summary The nature of Cuprolinic Blue-positive anionic filaments in mouse lung alveoli has been characterized. The contrast of filaments in the alveolar basement membrane of type I epithelial cells was lost on treatment with nitrous acid and pronase (without prefixation). In contrast, neither neuraminidase, chondroitinase ABC or AC, norStreptomyces hyaluronidase had any effect. Treatment with pronase (after prefixation) and 2.0m MgCl₂ (after prefixation) also had no effect, indicating that the filaments are heparan sulphate proteoglycans. The filaments in the alveolar basement membrane of type II epithelial cells and in the capillary basement membrane of the endothelial cells were also nitrous acid sensitive, but chondroitinase ABC-insensitive. A model in which the whole alveolus contains a single layer of heparan sulphate-containing proteoglycan monomers is proposed. Furthermore, the collagen fibril associated filaments remained unaffected after treatment with nitrous acid, neuraminidase orStreptomyces hyaluronidase, or after digestion with pronase (after prefixation) and treatment with 2.0m MgCl₂ (after prefixation). These filaments, however, could no longer be detected when digestion with chondroitinase ABC or pronase (without prefixation) was applied; chondroitinase AC treatment clearly affected the filaments, although they still were visible. These results indicate that the filaments are dermatan sulphate-containing proteoglycans. Some functional aspects of the proteoglycans are discussed.     

Electron microscopic observations on pulmonary connective tissue stained by Ruthenium Red

Summary Ultrastructural studies on human lung were performed with special attention to the interstitial acid mucopolysaccharides by Ruthenium Red staining and several enzyme digetion tests withStreptomyces hyaluronidase, chondroitinase ABC, chondroitinase AC, heparinase, trypsin and collagenase.Periodic lateral granules on the major cross bands of collagen fibrils and amorphous coats on them became visible by Ruthenium Red staining. The surface of elastic fibres, associated microfibrils, and some fine fibrils 10–20 nm in diameter were stained. Ruthenium Red also stained the surface of fibroblast and smooth muscle cells, basement membrane and filamentous long segments. In the interstructural space, granular substances 10–80 nm in diameter and fine filaments 3–4 nm thick, which formed a fine reticular network, were clearly observed. They were not visible on the usual thin section. The granular substances were located on the cross points of the fine filaments. They spread continuously and connected with each of the cells and extracellular structures in the pulmonary interstitium. The results of the enzyme digestion tests on the Ruthenium Red-positive material are discussed.     

Ultrastructural localization of proteoglycans in tissue using Cuprolinic Blue according to the critical electrolyte concentration method: Comparison with biochemical data from the literature

Summary Several connective tissues were stained for proteoglycans using the cationic dye Cuprolinic Blue according to the critical electrolyte concentration method. With this method, proteoglycans are visualized as electron-dense filaments. In most tissues, two types of proteoglycan filaments are present: a small (maximum length 60 nm), thin, collagen fibril-associated filament, and a thick, heavily-staining filament which is predominantly localized between bundles of collagen fibrils. Cartilage contains very large (about 300 nm) proteoglycan filaments while in cornea they are very small. Comparison with biochemical data from the literature suggests that the appearance of the proteoglycan filaments may be indicative for the glycosaminoglycan—protein ratio and for the molecular weight of the part of the protein core to which glycosaminoglycans are attached. The data thus obtained on the localization and structure of a proteoglycan may be useful when planning a strategy for its isolation.     

Cuprolinic Blue visualization of cytosolic and membrane-associated glycosaminoglycans in the rat junctional epithelium and gingival epithelia

Summary The gingiva of rat molars was studied at the light microscope level using glutaraldehyde as fixative, Cuprolinic Blue for visualizing polyanionic glycosaminoglycans and the autometallographic technique for enhancing the copper signal of the cationic dye. The polyanions were located inside the epithelial cells in the junctional epithelium, whereas a network located along either the plasma membrane or the intercellular spaces, or both, of the gingival oral epithelium and sulcular oral epithelium was evident with autometallography. In these cases, positive staining was limited to the basal and spinous layers, the granular and keratinized layers being unstained. With the transmission electron microscope, electron-dense aggregates were seen in the gingival lamina propria, in the basement membrane and along the plasma membrane of the keratinocytes of the basal and spinous layers of the gingival and sulcular oral epithelia. In the junctional epithelium, Cuprolinic Blue-positive granules, 25 nm in diameter, were seen in the cytoplasm. Together with some vesicles containing electron-dense material, they may account for the staining process noted after autometallography. When the ultra-thin sections were digested with bovine testicular hyaluronidase, the staining was abolished. This indicates that glycosaminoglycans were primarily responsible for the staining pattern visualized with these methods. In the junctional epithelium, the cytosolic location of the 25 nm granules reflects either transcellular transfer between the plasma membrane and the nucleus or accumulation of glycosaminoglycans in this group of keratinocytes. The glycoconjugates located inside vesicles or vacuoles are related to endocytosis and lysosomal degradation. Interstitial glycosaminoglycans seen in the two types of oral epithelium may play a role in the diffusion of water and nutriments.     

Differential staining of glycosaminoglycans in the predentine and dentine of rat incisor using Cuprolinic Blue at various magnesium chloride concentrations

Summary Rat incisors were fixed with a solution of 0.05% Cuprolinic Blue and 2.5% glutaraldehyde in the presence of various concentrations of MgCl₂ according to the critical electrolyte concentration (CEC) principle. This method allows glycosaminoglycans (GAG) to be properly preserved and visualized. Small granules were stained by the cationic dye in the predentine in the absence of MgCl₂. These granules grew in size and became more electron-dense when the concentration of the electrolyte was increased. Larger ribbon-like structures and granules were seen when 0.3M MgCl₂ was used. In the dentine, tiny dots in close association with the surface of the collagen fibres, or their periodic striations, were positively stained. A thick electron-dense band located on the dentine side at the predentine-dentine junction was seen both with and without 0.05 M MgCl₂. With higher concentrations of the electrolyte (0.1–0.3 M), this band was reduced to a very thin line located at the border of the dentine, along with mineralizing collagen fibres. This demonstrated the presence of GAG at the dentine surface and therefore indicated that GAG may play a role as nucleator agent.     

Further characterization of a large proteoglycan in human lung alveoli

By use of the cationic dye Cuprolinic Blue in a critical electrolyte concentration method, heavily staining, generally large, filaments have been demonstrated in human lung alveoli. In some lung specimens they are abundant, while in others they are very scanty. The filaments are seen: around bundles of collagen fibrils, at places which seem electron microscopically almost empty, associated with basement membranes around elastin, and sometimes associated with individual collagen fibrils. After poststaining tiny threads--connecting the filaments--could sometimes be observed. The filaments are resistant to treatment with nitrous acid, heparitinase or pronase after prefixation. After digestion with chondroitinase ABC, chondroitinase AC or pronase without prefixation, the filaments are no longer detectable. The tiny threads are chondroitinase ABC resistant. It is concluded that the Cuprolinic Blue-positive filaments represent proteoglycans which contain chondroitin sulfate and/or glucuronic acid-rich dermatan sulfate. The possible role of these proteoglycans in tissue repair is discussed.     

The proteoglycan skeleton of the Kurloff body as evidenced by Cuprolinic Blue staining

Summary This study deals with the ultrastructure of the chondroitin sulphate proteoglycans of the Kurloff body, a large lysosome organelle, metachromatic towards Toluidine Blue, of a blood cell unique to the guinea pig and called the Kurloff cell. Splenic Kurloff cell from oestrogen-treated guinea pig cells were examined after staining with Cuprolinic Blue, a cationic phthalocyanine-like dye, in the presence of MgCl₂ in a critical electrolyte concentration method. Better results were obtained when the fixation-staining by the glutaraldehyde Cuprinolinic Blue MgCl₂ mixture was preceded by a glutaraldehyde pre-fixation. On light microscopy, Kurloff bodies generally exhibited an overall pink and glassy metachromasia, sometimes with additional darker metachromatic small dots at their peripheries. At the ultrastructural level, the metachromatic central matrix of the Kurloff body usually exhibited, as a major feature, a typical network pattern of ribbon-like or stellate electron-dense precipitates suggesting the presence of a skeleton of Cuprolinic Blue-reactive filamentous structures. Taking into account their high anionicity (as shown by the stability of the dye binding in the presence of 0.3 m MgCl₂) and their susceptibility to chondroitinase ABC, these anionic structures were assumed to be related to the proteochondroitin-4-sulphate previously characterized as the only major sulphated glycoconjugate of the Kurloff cell.     

Cuprolinic Blue: a specific dye for single-stranded RNA in the presence of magnesium chloride. I. Fundamental aspects

Summary Qualitative and quantitative aspects of the cationic dye Cuprolinic Blue were investigated with model films of polyacrylamide gel in which RNA, DNA and other biological polyanionic compounds had been incorporated. In the presence of 1m MgCl₂, Curpolinic Blue was found to bind specifically to single-stranded RNA, leaving native DNA, proteins, (acid) polysaccharides and phospholipids completely unstained. Under these conditions, Cuprolinic Blue is complexed by non-electrostatic bonds with non-stacked purine bases, mainly adenine. Optimal conditions for dye binding and differentiation have been defined. Both the Cuprolinic Blue-MgCl₂ staining of single-stranded RNA and the Cuprolinic Blue staining of RNA and DNA in the absence of MgCl₂ were found to obey the Lambert-Beer law. The advantages and possible applications of Cuprolinic Blue are compared with well-known (indirect) histochemical RNA staining procedures.     

Electron microscopic visualization of proteoglycans with Alcian Blue.

The intercellular matrices of bovine nasal cartilage, chick embryo perichordal cartilage, and chick embryo mesenchymal cells cultured in vitro have been examined by electron microscopy after staining them with Alcian Blue in salt solutions according to Scott & Dorling (1965). Matrix granules, which are typical components of cartilage at the ultrastructural level, are not visible after Alcian Blue staining and are replaced by alcianophilic rod-like particles, varying in length and width. With tissue cultures, Alcian Blue stains 40-120 A thick filaments which display an orthogonal and longitudinal relationship to collagen fibrils. We assume that cartilage matrix granules represent linear proteoglycans that are coiled as a consequence of the usual glutaraldehyde-osmium fixation. It is thought that Alcian Blue, on the other hand, contributes to the stabilization of the proteoglycans in their original structural arrangement. This stabilizing property presumably also results in the sharp visualization of fine filaments in the tissue culture matrix.     

Cationic dyes reveal proteoglycans on the surface of epithelial and endothelial kidney cells

Summary The glomerular epithelial cells of the rat kidney fixed by vascular perfusion with an aldehyde solution containing either safranine O or alcian blue (and 0.3 M MgCl₂) display filaments which are located close to the outer surface of the plasma membrane. These filaments are similar to those revealed by the same methods in the laminae rarae of the glomerular basement membrane. Alcian blue (and MgCl₂) further demonstrates the presence of anionic sites inside the endothelial cell pores of the glomerular and peritubular capillaries, on the luminal surface of endothelial cells of large renal vessels and along the basolateral surface of the epithelial cells of the Bowman capsule and of the proximal convoluted tubule.Supported in part by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 146)     

Glomerular anionic charge in congenital nephrotic syndrome of the Finnish type

Summary Decrease of the anionic charge of the glomerular basement membrane and especially the reduced amount of heparan sulphate proteoglycan in the lamina rara externa has been suggested to be the basic pathogenetic defect in congenital nephrotic syndrome. In the present study the anionic charge of glomeruli was examined in the congenital nephrotic syndrome of the Finnish type and in controls using cationic stains (polyethyleneimine, Ruthenium Red) in electron microscopy. Chondroitinase and heparinase treatments were used to characterize further the anionic elements detected. Scanning electron microscopy (SEM) was used in addition to transmission electron microscopy (TEM) to examine the tridimensional structure and secondary changes of podocytes in this syndrome. The number (mean ± SD) of polyethyleneimine granules per 1 μm length of lamina rara externa of the glomerular basement membrane was 24.9 ± 4.5 in control and 2.32 ± 4.3 in congenital nephrotic syndrome subjects. The Ruthenium Red staining pattern was closely similar in syndrome and control kidneys. The granules evident after staining with either cationic stain were seen after chondroitinase but not after heparinase treatment in control as well as in syndrome patient kidney samples. No denuded areas of basement membrane in 42 glomeruli from four syndrome patients were found in SEM. In conclusion, the amount of anionic sites in the lamina rara externa as detected by either cationic stain was comparable to controls. These results do not support the hypothesis of decreased anionic sites in the lamina rara externa of the glomerular basement membrane in congenital nephrotic syndrome of the Finnish type.     

Glycosaminoglycans in porcine lung: an ultrastructural study using Cupromeronic Blue

Glycosaminoglycans (GAGs) are essential components of the extracellular matrix contributing to the mechanical properties of connective tissues as well as to cell recognition and growth regulation. The ultrastructural localization of GAGs in porcine lung was studied by means of the dye Cupromeronic Blue in the presence of 0.3 M MgCl₂ according to Scott's critical electrolyte concentration technique. GAGs were observed in locations described as follows. Pleura: Dermatan sulphate (DS) and chondroitin sulphate (CS) attached in the region of the d-band of collagen fibrils, interconnecting the fibrils; heparan sulphate (HS) at the surface of elastic fibers and in the basement membrane of the mesothelium and blood vessels. Bronchial cartilage: Abundant amounts of GAGs were observed in three zones: pericellular, in the intercellular matrix and at the perichondrial collagen. By enzyme digestion a superficial cartilage layer with predominantly CS could be distinguished from a deep zone with CS and keratan sulphate. The structure of the large aggregating cartilage proteoglycan was confirmed in situ. Airway epithelium: HS at the whole surface of cilia and microvilli and in the basement membrane of the epithelial cells. Alveolar wall: CS/DS at collagen fibrils, HS at the surface of elastic fibers and in the basement membranes of epithelium and endothelium.     

Arrangement of connective tissue components in the walls of seminiferous tubules of man and monkey.

In primates the membrane separating the seminiferous epithelium from the interstitial space is composed of one to three (monkey) or two to six layers (man) of myoid cells associated with one to two layers of fibrocyte-like adventitial cells. All these cells are separated from each other by irregular spaces filled with various connective tissue intercellular components. Subjacent to the elements of the seminiferous epithelium is a continuous, often redundant, basement membrane. A similar basement membrane-like material forms a layer next to and over small areas of the plasma membrane of myoid cells. Collagen fibrils grouped in bundles of various sizes are seen in all connective tissue layers but are particularly abundant in the space between the seminiferous epithelium and the innermost layer of myoid cells. Elastic fibrils demonstrated by the Verhoeff iron hematoxylin technique are also present. Composed of a homogeneous material, the elastic fibrils are short, irregular, branching entities with a diameter comparable to or smaller than that of collagen fibrils. In addition, an abundance of microfibrils with a diameter of 12-15 nm is present in the various connective tissue layers. These microfibrils have a densely stained cortex and a lightly stained core. When seen close to the myoid cells, bundles of micro fibrils appear to insert on well defined areas next to the plasma membrane. These areas commonly face the patches of electron-dense material observed on the inner aspect of the plasma membrane of the myoid cells and in which the actin filaments are inserted. Bundles of microfibrils often span the gap between myoid cells of the same layer as well as those of adjacent layers. Microfibrils are also closely related to the surface of elastic fibrils and are seen intertwining with collagen fibrils. Thus microfibrils appear to bridge and bind together adjacent myoid cells and anchor the surface of these cells to the bundles of elastic and collagen fibrils present in the intercellular spaces of the limiting membrane.     

Particular structure of the anterior third of the human true vocal cord.

The histological aspects of the true vocal cord mucosa change in the anterior third compared with the posterior two thirds. The anterior third is characterized by an epithelium where the ridges, marked in the posterior two thirds, are very slight or even absent. The underlying basement membrane, which is thin in the posterior two thirds, here appears particularly thick. At the ultrastructural level in this area, beneath a normally thickened basal lamina, a thick layer of finely granulated electron-dense material, interspersed with thin and randomly scattered collagen fibrils and proteoglycan filaments, is detectable. Beneath this thickened basement membrane, a layer of small undulated collagen fibril bundles with very numerous interspersed oxytalan fibres is found. The collagen fibrils, small in diameter (30-40 nm), seem to continue with the collagen fibrils of the basement membrane. In this layer numerous blood vessels with a very thick, delaminated basement membrane are also observed. The underlying area is characterized by the vocal cord ligament, composed by large compact collagen fibril bundles with interspersed elastic fibres. The particular features of the thick basement membrane, the thick-walled and delaminated vessels and the modular distribution of the elastic system together may well form the basic structure enabling the functional integration of the vocal ligament into the overlying mucosa and the underlying vocal muscle.     

Ruthenium red staining and tannic acid fixation of dental basement membrane

Summary Ruthenium red staining and tannic acid fixation were used to analyse the fine structure of embryonic mouse dental basement membrane in intact first mandibular molars or in EDTA-isolated dental papillae. Preameloblasts are separated from extracellular matrix proper by a basal lamina that contains regularly arranged proteoglycan granules of about 10 nm in diameter. This distribution pattern is particularly evident in the inner and outer lamina rara of the basal lamina associated with EDTA-isolated dental papillae. The plasmalemma of preameloblasts demonstrates electron dense plaques on the inner leaflet. Ruthenium red positive granules (50 nm in diameter) coat non-striated and striated fibrils of the matrix. Hyaluronidase treatment digested the ruthenium red positive granules. Tannic acid fixation allowed the demonstration of filaments within the lamina rara interna, connecting the lamina densa with plasmalemma of preameloblasts. These observations are discussed in the context of the terminal differentiation of odontoblasts.These studies were supported by INSERM, grant n 537785 and DGRST     

Ultrastructure of proteoglycans in the specific granules of guinea-pig basophilic leukocytes as demonstrated by cuprolinic blue staining

The ultrastructure of sulphate proteoglycans in basophil granules was examined using cytochemical procedures designed to stabilize and visualize these highly anionic macromolecules in situ. Unfixed or glutaraldehyde-prefixed guinea-pig spleen cells were submitted to fixation/staining in 2.5% glutaraldehyde, 0.2% cuprolinic blue (CB; a cationic phthalocyanin dye) and 0.2 or 0.3M MgCl₂ with or without glycosidase treatments. Abundant electron-dense precipitates were present throughout the granule matrix. The stained structures were often arranged in a quasi-crystalline typical banded pattern. Negative control basophils had no electrondense precipitates. Digestion with chondroitinase ABC destroyed the CB-positive electron-dense banded or filamentous patterns while sialidase treatment did not, but led to larger CB-positive filaments in the cytoplasm near the granules. Taking into account their high anionicity, as shown by the stability of dye binding in the presence of 0.3M MgCl₂, and their susceptibility to chondroitinase ABC, the CB-precipitates are assumed to be related to the sulphated proteoglycans previously characterized in basophil granules. The CB-positive crystalline or filamentous network of the granule matrix is also assumed to reflect the in situ location and organization of these intracellular proteoglycans and may be involved in maintaining the shape of the granule.     

Electron microscopic autoradiography of 35SO4-labelled material closely associated with collagen fibrils in mammalian synovium and ear cartilage.

Synopsis Electron microscopic autoradiography of connective tissue obtained from mice and rabbits previously injected with³⁵SO₄ indicated that sulphated proteoglycans are localized on collagen fibrils. Ruthenium Red-positive transverse belts surrounding fibrils near the a-bands were heavily labelled, but fine lateral filaments of Ruthenium Red-positive material were not. These filaments, which interconnect collagen fibrils in a variety of connective tissues may represent linear aggregations of hyaluronic acid, glycoproteins and non-sulphated or long-lived sulphated proteoglycans.     

Connective tissue of the livers of newborn and adult marmosets (Callithrix jacchus)

Immunofluorescence microscopic and electron microscopic investigations revealed components of the matrix and of the basal lamina (collagen type I, III, IV and V, BL-heparan sulfate and fibronectin) in the sinus wall (Disse's space) of the livers of newborn and adult marmosets (Callithrix jacchus). Collagen type I was missing in both the two age groups. Small amounts of laminin were present in the livers of newborn and absent in those of adult animals, whereas collagen type III occurred in the form of delicate fibres. Light microscopic inspection showed a continuous distribution of all other components in the sinus wall. The amount of collagen type III and V increased depending on the age. Electron microscopic investigations revealed single or bundled fibrils (20-30 nm) and filaments (10-12 nm). After addition of tannic acid, plaques of a fine-filamentous network and incorporated granules were observed. After addition of resting Ruthenium Red, electron-dense granules (20-60 nm) were irregularly distributed in the structureless space, resting on collagenous fibrils and cell membranes. The fibrils were allocated to collagen type III, the filaments to collagen type V. The plaques were supposed to contain heparan sulfate, collagen type IV and fibronectin. The absence of a Lamina densa of the basal lamina was attributed to the absence of laminin which probably plays an important role in the formation of this layer. Differences in the distribution pattern of the matrix components and thus a functional mosaic of the permeability of Disse's space were assumed. The complete absence of collagen type I and laminin in the lobules makes the adult marmoset liver especially suited for studies on the importance of this collagen type under pathological conditions, since both components are expressed in this way.     

Basement membrane-like matrix of teratocarcinoma-derived endodermal cells: presence of laminin and heparan sulfate in the matrix at points of attachment to cells

Teratocarcinoma-derived endodermal PYS-2 cells are known to synthesize an extracellular matrix containing the basement membrane molecules laminin, type IV collagen, and heparan sulfate proteoglycan as major constituents (I. Leivo, K. Alitalo, L. Risteli, A. Vaheri, R. Timpl, J. Wartiovaara, Exp Cell Res 137:15-23, 1982). Immunoferritin techniques with specific antibodies were used in the present study to define the ultrastructural localization of the above constituents in the fibrillar network. Laminin was detected in matrix network adjacent to the basal cell membrane and in protruding matrix fibrils that connect the matrix to the cell membrane. Ruthenium red-stainable heparinase-sensitive 10- to 20-nm particles were often present at the junction of the attachment fibrils and the matrix network, or along the attachment fibrils. A corresponding distribution of ferritin label was observed for basement membrane heparan sulfate proteoglycan. Type IV collagen was found in the matrix network but not in the attachment fibrils. The results suggest that the PYS-2 cells are connected to their pericellular matrix by fibrils containing laminin associated with heparan sulfate-containing particles. These results may also have relevance for the attachment of epithelial cells to basement membranes.