Pericellular Area Research Articles

Synergistic effect of BMP-2 and ascorbate on the phenotypic expression of osteoblastic MC3T3-E1 cells.

Of TGF-beta superfamily proteins, BMP-2 enhanced alkaline phosphatase (ALP) activity in cultured osteoblastic cells, MC3T3-E1, to the same level promoted by ascorbate, whereas TGF-beta s (beta 1, beta 2, beta 3) reduced ALP activity and altered cell morphology under the same conditions. Activin appeared to have no distinct effect on ALP synthesis. Ascorbate dependent increase in ALP synthesis was markedly stimulated in the presence of BMP-2. The synergistic effect of ascorbate on ALP synthesis was replaced by type I collagen coated on the culture dish. However, BMP-2 appeared not to bind to type I collagen. These findings indicate that BMP-2 acts directly on osteoblastic cells through its receptors and collagenous matrix can neither recruit BMP-2 nor modulate directly the action of BMP-2 in the pericellular area. Treatment of cells grown in ascorbate media with TGF-beta s decreased rapidly the cellular ALP activity indicating that TGF-beta s direct cells to the dedifferentiated stage.

Immunohistochemical study on collagenous proteins and biophysical analysis of crystals in extraskeletal chondroma.

The immunohistological distribution of collagen types I, II, III, and VI in five cases of extraskeletal chondroma was examined and compared with that in six cases of enchondroma. In addition, the composition of crystals deposited in three cases of extraskeletal chondroma were biophysically analyzed with special attention to the relationship between the collagen types of the matrix and the crystal deposition. In extraskeletal chondroma, immunoreactivity of type II collagen in the extracellular matrix and type VI collagen in the pericellular area, which were strongly and diffusely recognized in the normal hyaline cartilage and enchondroma, was diminished. Instead, additional types of collagen, types I and III, were demonstrated in the matrix. Electron roentgenographic microanalysis and infrared light spectroscopic analysis revealed that calcium pyrophosphate dihydrate (CPPD) was included in the crystals of extraskeletal chondroma. CPPD crystals were observed in/around collagen types I and III. The possible relationship between the difference of collagen composition in the matrix and the CPPD crystal deposition is discussed.

Tenascin distribution in articular cartilage from normal subjects and from patients with osteoarthritis and rheumatoid arthritis.

To determine whether tenascin is present in normal and diseased human cartilage. Immunohistochemical and biochemical assays with a monoclonal antibody against all tenascin isoforms (BC-4) were used. Cartilage samples from osteoarthritis and rheumatoid arthritis patients contained increased amounts of tenascin compared with the levels in normal cartilage. Human fetal cartilage was also found to contain tenascin. In normal cartilage explants treated with interleukin-1 beta, tenascin was present in pericellular areas of all layers. Immunolocalization studies revealed that tenascin was most abundant in the superficial layers of osteoarthritic cartilage. Western blot analysis performed from dissociative extracts of diseased cartilage confirmed the presence of subunits of the native molecule. Tenascin is increased in arthritic cartilage and is weakly expressed in normal cartilage.

Localization of tenascin in human skin wounds--an immunohistochemical study.

A total of 56 surgically treated human skin wounds with a wound age between 8h and 7 months were investigated. Tenascin was visualized by immunohistochemistry and appeared first in the wound area pericellularly around fibroblastic cells approximately 2 days after wounding. A network-like interstitial positive staining pattern was first detectable in 3-day-old skin wounds. In all wounds with an age of 5 days or more, intensive reactivity for tenascin could be observed in the lesional area (dermal-epidermal junction, wound edge, areas of bleeding). In wounds with an age of more than approximately 1.5 months no positive staining occurred in the scar tissue. In conclusion, for forensic purposes, positive staining for tenascin restricted to the pericellular area of fibroblastic cells indicates a wound age of at least 2 days. Network-like structures appear after approximately 3 days or more. Since tenascin seems to be regularly detectable in skin wounds older than 5 days, the lack of a positive reaction in a sufficient number of specimens indicates a wound age of less than 5 days. The lack of a positive reaction in the granulation tissue of wounds with advanced wound age indicates a survival time of more than about 1.5 months, but a positive staining in older wounds cannot be excluded.

Scanning Electron Microscopy of Proteoglycans in Metaphyseal Cartilage

Alcian blue (AB) was used for scanning electron microscope investigations on metaphyseal cartilage. In the pericellular area three-dimensional network connecting the cell membrane surface to the lacunar wall is evident. The network is formed by very long rod-like filaments about 50 nm thick. The segments may be interpreted as the proteoglycans (PGs) of the pericellular area. In the pericellular area in hypertrophic and degenerative zones, the rod-like segments are closely connected to "chain granules" which are the morphological expression of Ca-P non crystalline compounds. The rod-like segments are not at all evident either in glutaraldehyde-osmium fixed fragments or in predigested-(streptococcal hyaluronidase and chondroitinase) AB stained ones. It is concluded that AB is a good method to detect the three-dimensional spatial disposition of cartilage PGs.

The expression and localization of urokinase-type plasminogen activator and its type 1 inhibitor are regulated by retinoic acid and fibroblast growth factor in human teratocarcinoma cells.

Human Tera 2 embryonal carcinoma cells switch gradually from rapidly growing undifferentiated cells to almost nonproliferating cells during retinoic acid (RA)-induced neuronal differentiation. This process is associated with the increased expression of type 1 plasminogen activator inhibitor (PAI 1) mRNA, and the secreted inhibitor is immobilized to the pericellular area. Furthermore, the differentiation is accompanied by a decrease in the amount of both the secreted tissue-type PA (tPA) and the mainly cell-associated urokinase-type PA (uPA) activity. In RA-differentiated cells, uPA becomes localized at the vinculin-rich cell-substratum adhesion sites. Fibroblast growth factor activity has been associated with various events during embryonal growth and with the regulation of proteolytic enzymes. A short-term treatment of the undifferentiated Tera 2 cells with basic fibroblast growth factor (bFGF) increases uPA mRNA levels and the cell-associated uPA activity, whereas the secretory tPA activity decreases. bFGF induces PAI 1 mRNA expression in the undifferentiated cells, but unlike PAI 1 protein after RA-treatment, the inhibitor does not accumulate around the cells but is released in the medium. A similar exposure to bFGF has less effect on the RA-differentiated Tera 2 cells. Under these conditions bFGF treatment leads to an increase in the amounts of PAI 1 and uPA mRNAs, but no changes in the localization of these components can be seen. Differentiation of human embryonal carcinoma cells is thus connected with an altered response to bFGF.

Enzymatic degradation of tumor necrosis factor by activated human neutrophils: Role of elastase

Although the role of tumor necrosis factor-alpha (TNF) as mediator of inflammation is now well established, its interactions with polymorphonuclear neutrophils (PMN) are not fully understood. Therefore, we investigated the possible hydrolytic action on TNF of intra-lysosomal enzymes released by activated PMN in the extracellular medium. We first incubated 125I radiolabeled TNF in vitro with activated PMN and by HPLC analysis, we observed a degradation process completely blocked by the previous addition of alpha 1-Antitrypsin (AT) to the incubation medium. By comparing several degradative patterns of TNF obtained with purified leukocyte proteases and supernatant of activated PMN, we identified elastase as the major enzyme involved in this catabolic process of TNF. In a second part, we determined the bioactivity of the cleavage fragments of recombinant human TNF (rhTNF) by a cytotoxicity assay. None of the fragments was found biologically active. Our results suggest that, at inflammatory sites, an enzymatic degradation of TNF may occur in the pericellular area of activated PMN. This new catabolic pathway leading to inactivation of TNF might be regarded as an effective local negative feed-back process limiting the potentially toxic effects of this cytokine.

Application of the ferrocyanide-reduced osmium method for mineralizing cartilage: Further evidence for the enhancement of intracellular glycogen and visualization of matrix components

The ferrocyanide-reduced osmium (FRO) fixation method was applied to neonatal mouse mandibular condylar cartilage for its processing for electron microscopy. The results were compared to those obtained by the conventional glutaraldehyde-osmium tetroxide fixation method. Three different stages in the life cycle of condylar cartilage cells were examined. FRO enabled the visualization of delicate fibrillar mesh in the matrix of all three zones of the cartilage, resulting in a dense appearance of the intercellular matrix. The classical stellate shape of matric granules seen in cartilage fixed with glutaraldehyde-osmium tetroxide was not observed in FRO-processed tissues. Chondrocytes that were FRO-processed almost entirely filled their lacunar space. In their pericellular area, fibrillar material and electron-dense aggregates could be demonstrated by the FRO method. As a conclusion of this study, it is recommended to supplement a conventional protocol with the FRO fixation method for routine and research purposes.

Immunoelectron microscopic localization of fibronectin in the smooth muscle layer of mouse small intestine.

We studied the ultrastructural distribution of fibronectin in the smooth muscle layer of mouse small intestine with affinity-purified antibodies using the immunogold technique. Fibronectin was present over the pericellular area extending from the cell membrane to the extracellular matrix beyond the basal lamina. Distribution of the glycoprotein over the pericellular area was heterogeneous, i.e., it was localized more abundantly in the narrow space between smooth muscle cells, the gaps having a width of 60-80 nm where the two dense bands in adjacent cells matched each other. Such localization suggests that fibronectin contributes to cell adhesion. Within the basement membrane, gold label was localized both in lamina lucida and lamina densa, more densely in the latter than in the former. Fibronectin was also co-distributed with collagen fibers in the extracellular matrix. Within smooth muscle cells, gold particles were observed on rough endoplasmic reticulum and secretory vesicle-like structures. These results suggest that smooth muscle cells synthesize fibronectin and secrete it as a component of the basal lamina and extracellular matrix.

Normal and malignant cells, including neurons, deposit plasminogen activator on the growth substrata

The results of four different assay methods showed that both normal and malignant plasminogen activator-secreting cells deposited substantial amounts of this protease on tissue-culture substrata, including collagen coatings. The cells studied were Rous sarcoma virus (RSV)-transformed vole fibroblasts, a malignant neural cell line (NG108-15) capable of neurite formation, and normal mouse-regenerating sensory neurons. Deposited plasminogen activator was detected by a fibrin overlay assay at sites from which cells growing on coverslips had been gently dislodged, showing that active enzyme is left beneath cells and in the immediate pericellular area. For neuronal cells, fibrinolytic zones were detected not only at the previous positions of cell bodies but also along the terrain conditioned by neurite extension, suggesting that a trail of plasminogen activator is left behind during growth cone movement. Substratum-bound enzyme could be solubilized in buffers containing sodium dodecyl sulfate (SDS) or Triton X-100 and demonstrated by zymography following electrophoresis or assayed for amidolytic activity with a chromogenic substrate (Kabi S-2251). The results suggest that plasminogen activator may be considered a component of substrate-adhesion material. Secretory proteases deposited directly on matrix molecules would seem strategically positioned to participate in local degradation of components of the extracellular environment.

Cell configuration and adhesive properties of metastasizing and non-metastasizing BSp73 rat adenocarcinoma cells

The pattern of cell substrate interaction, the cell surface composition and the organization of cytoskeletal elements was studied in tumour cell variants of the BSp73 rat adenocarcinoma displaying different metastatic capabilities and cell configuration. The non-metastasizing AS variant cells adhered to the substrate and spread via vinculin-containing focal contacts. These cells also synthesized, secreted and assembled fibronectin at the pericellular area. The metastasizing ASML variant cells adhered to the substrate at a slower rate via thick cytoplasmic protrusions, but were removed from the substrate by trypsin-EDTA slower than the non-metastasizing AS variant cells. The ASML cells also synthesized very low levels of both vinculin and fibronectin, displayed a diffuse pattern of actin and tubulin organization, and were unable to spread on the substrate. Spreading could not be induced in the ASML cells by seeding the cells on an extracellular matrix derived from bovine corneal endothelial cells or on concanavalin A (conA)-coated substrates, or by the addition of db-cAMP to the medium. The metastasizing cells expressed a unique and abundant cell surface glycoprotein of M r 170 000 which was also shedded into the growth medium. The relationships among the adhesive properties, the organization of cell surface components and of the cytoskeleton in the tumour cell variants, and the expression of their metastatic phenotype is discussed.

The immunohistochemical distribution of collagens type IV, V, VI and of laminin in the human oral mucosa

The distribution of collagens type V (form AB 2) and VI was investigated on cryostat sections of normal human oral mucosa by indirect immunofluorescence. For comparison, antibodies to fragments of type IV collagen and laminin were also used to delineate basement membrane containing structures. All antibodies used were raised against human proteins. Type V collagen appeared as a microfibrillar structure throughout the interstitium, apparently touching but not being present within epithelial or vascular basement membranes. Microfibrils in blood vessel walls were limited to the intimal layer. Pericellular areas were not specifically stained. Type VI collagen appeared as an almost amorphous stromal structure becoming more prominent and more fibrillar in the upper connective tissue papillae. Intense staining was observed in the media of blood vessels and around smooth muscle cells. A possible role of type VI collagen in tissue stabilization may be expected from this ubiquitous and abundant distribution. The findings identify types V and VI collagen as important structures in the oral mucosa and serve as a basis for understanding morbid changes.

Association of glycoconjugates with the cytoskeletal framework.

The association of glycoconjugates with the cytoskeletal framework was examined in detergent-extracted cells. Sparse cultures of fibroblasts that assemble only minimal amounts of extracellular matrix were extracted under mild conditions with Triton X-100 which remove most of the lipids and soluble cellular proteins. The detergent-resistant framework retains lectin binding sites in the nucleus, in the perinuclear area occupied by the rough endoplasmic reticulum-Golgi system of the intact cell, and in a network throughout the cytoskeletal framework. Fluorescent-antibody staining with antibody against collagen type I and fibronectin reveals extensive perinuclear staining of the remnant rough endoplasmic reticulum-Golgi system. In contrast, only sporadic staining of the pericellular area is obtained with these antibodies, in sparse cultures of whole cells. Lectin binding sites were detected in the nucleus and are attributed to chromatin-associated glycoconjugates. They can be removed by DNase under conditions that preserve the cytoplasmic lectin binding sites and the nuclear matrix. The results suggest a high degree of integration of the membrane residues of the cytoplasmic elements and the nuclear matrix with the skeletal framework and indicate a possible role for the glycoconjugates in this structural integration.

Association of glycoconjugates with the cytoskeletal framework.

The association of glycoconjugates with the cytoskeletal framework was examined in detergent-extracted cells. Sparse cultures of fibroblasts that assemble only minimal amounts of extracellular matrix were extracted under mild conditions with Triton X-100 which remove most of the lipids and soluble cellular proteins. The detergent-resistant framework retains lectin binding sites in the nucleus, in the perinuclear area occupied by the rough endoplasmic reticulum-Golgi system of the intact cell, and in a network throughout the cytoskeletal framework. Fluorescent-antibody staining with antibody against collagen type I and fibronectin reveals extensive perinuclear staining of the remnant rough endoplasmic reticulum-Golgi system. In contrast, only sporadic staining of the pericellular area is obtained with these antibodies, in sparse cultures of whole cells. Lectin binding sites were detected in the nucleus and are attributed to chromatin-associated glycoconjugates. They can be removed by DNase under conditions that preserve the cytoplasmic lectin binding sites and the nuclear matrix. The results suggest a high degree of integration of the membrane residues of the cytoplasmic elements and the nuclear matrix with the skeletal framework and indicate a possible role for the glycoconjugates in this structural integration.

The measurement of proteoglycan in the mineralizing region of the rat growth plate.

Using a recently developed technique for measuring proteoglycan by x-ray microprobe analysis, we examined six-week-old rat growth plates. In the proliferating zone, the proteoglycan concentration was found to be relatively low in the transverse septa, but somewhat higher in the longitudinal septa between the proliferating chondrocytes. In the lower hypertropic zone, in the region of the degenerating hypertrophic chondrocytes, the immediate pericellular area had a very high concentration of proteoglycan, as was obtained. These results indicate that either proteoglycan concentrations fall to very low levels in fully mineralized cartilage, or that the mineralization of cartilage interferes in some way with the measurement of microprobe signals from proteoglycans. We speculate that high levels of proteoglycan are necessary for mineralization in the epiphysis of the rat. The spot-to-spot analysis of proteoglycan by microprobe demonstrates the usefulness of this tool in analyzing minute volumes of cartilage for proteoglycan. It further demonstrates that mineralization of growth-plate cartilage is associated with changing patterns of concentration of proteoglycan. These normal patterns may constitute a basis for comparison in similarly evaluating concentrations of proteoglycan in diseases of growth disturbance.

Staining of glycosaminoglycans of intervertebral disc tissue

Intervertebral discs of six species were stained with Alcian blue and PAS methods to identify glycosaminoglycans. The polyanions are most concentrated in 'ring-like' pericellular areas and in material lying between individual lamellae and bundles of collagen. A slight increase occurs early in life and towards the disc centre. The concentration of glycosaminoglycans cannot be correlated with species which are known to suffer from clinical disc disease, though there appears to be a higher concentration in the discs of the cat and dog than in other species studied.

Simultaneous localization of proteoglycan by light and electron microscopy using toluidine blue O. A study of epiphyseal cartilage.

The simultaneous localization of proteoglycan by light and electron microscopy was demonstrated by fixing epiphyseal cartilage in a glutaraldehyde toluidine blue O solution. Sections cut for light microscopy viewing and those cut for electron microscopy required no further staining, although, in the latter case, staining with uranyl acetate and lead improved the overall contrast. By this technique, electron-dense structures were seen concentrated about the cells which were actively synthesizing matrix, and these structures appeared to bind collagen fibrils. Similar structures were not seen in conventionally fixed tissue. They could also not be identified when the specimens were previously incubated with the proteoglycan-digesting enzyme, papain, prior to toluidine blue O fixation. The toluidine blue O fixation method, unlike conventional fixation and staining, retained proteoglycan in the pericellular areas of actively synthesizing cells and made it visible by light and electron microscopy. It appears that proteoglycans is both precipitated and stained by the presence of toluidine blue O during fixation.

HISTOCHEMICAL STUDIES ON THE DISTRIBUTION OF ADENOSINE TRIPHOSPHATASE IN THE TRIGEMINAL GANGLION CELLS OF THE RAT

A detailed histochemical study of the distribution of adenosine triphosphatase among trigeminal ganglion cells, associated nerves and epi-, peri-, and endoneurium has been carried out. The latter as well as the pericellular zones show the highest degree of the enzymatic activity. The intracellular activity, although feeble in most of the cases as compared to the pericellular zones, is revealed as morphological entities—as filamentous or vesicular bodies or solid clumps. These are sometimes arranged in the perinuclear zone, at other times as concentric rings in the middle part of the cytoplasm. They may be concentrated at the peripheral part or distributed as isolated bodies throughout the cytoplasm. It has been suggested that these bodies, distributed in various topographical fashions, represent the enzymatically active site of the endoplasmic reticulum. Further, a coordination of various stages seen among different cells, suggests that there is a shifting of the enzymatic activity from the perinuclear zone to the peripheral part of the cytoplasm. At the latter site and in the adjacent pericellular areas there is a strong possibility of an enzymatic role in the membrane permeability. The enzyme may also be playing such a role in epi-, peri- and endoneurium.