Stereoscopic scanning electron microscopy of the red pulp of dog spleen with special reference to the terminal structure of the cordal capillaries

Summary In order to obtain direct morphological information about the three-dimensional fine structure of the splenic terminal vascular bed, especially the terminating mode of the cordal capillaries, stereoscopic scanning electron microscopy of perfusion-fixed and freeze-fractured red pulp of a normal dog spleen was undertaken.An improved method of perfusion-fixation was utilized in which the hydrostatic pressures of the splenic artery and vein were maintained at approximately the same levels as those in the living state. Stereoscopic observations of scanning electron micrographs clearly demonstrated the three-dimensional fine architecture of the splenic sinuses, the spongy cordal reticular tissue and the intracordal vasculature.The cordal capillaries terminate in the labyrinthine cordal space according to a certain mode in which the walls of the terminals are transformed into a meshwork structure continuous with the cordal reticular tissue owing to an increase in number and size of fenestrations. No evidence could be detected to prove or suggest any direct continuity of the capillary end with the splenic sinus. These results strongly support the concept of an open circulation, at least in the red pulp of the dog spleen, with the possibility of a functionally closed circulation under some physiological conditions.     

Freeze-fracture investigation of the red pulp of human spleen

This study concerns the morphology of the human spleen in freeze-fracture replicas and compares this with the findings in ultrathin sections. The material investigated consisted of two spleens resected at gastrectomy and one resected because of splenomegaly in a case of hairy cell leukaemia. The current concepts concerning the ultrastructure of the spleen were generally confirmed with the freeze-fracture technique. The sinus walls were found, as expected, to consist of closely fitting endothelial cells, which were identifiable in freeze-fracture replicas by numerous caveolae of the cell membrane. Contrary to the opinion upheld in the literature, the sinus endothelial cells were occasionally found to be connected by desmosomes or maculae adhaerentes. Corresponding to the finding of desmosomes in ultrathin sections, focal collections of intramembranous particles were observed in freeze-fracture replicas and a positive immunohistochemical reaction for desmoplakin in the sinuses was found at the light microscopic level. The view generally held in the literature that sinus endothelial cells can exhibit tight junctions was not confirmed. However, such junctions were found between vascular endothelial cells. The ring fibres of the sinuses, which are closely connected to the sinus endothelial cells through contractile fibres, apparently have various functions. Firstly, they contribute towards maintaining mechanical stability. Secondly, they represent basement membranes through which exchange occurs between the sinus endothelial cells and their surroundings. This is indicated by the caveolae and vesicles that are often found here in large numbers and in focal collections. Hairy cells exhibit no features in freeze-fracture replicas to suggest a cytogenetic relationship to interdigitating reticulum cells.     

The specific heat of red cells,with special reference to the paracrystalline rat red cell

The specific heat of the rat red cell, kept in cold sodium citrate, changes in the neighborhood of 6°C., the temperature near which the cell passes from its paracrystalline state to a state of greater disorder. The change in the specific heat is from 0.74 with a standard deviation of ±0.022 (paracrystalline state) to 0.87 with a standard deviation of ±0.021 (normal state). Although it has been looked for, no evidence of a change in specific heat has been found, between 1°C. and 15°C., in the case of the human red cell or of the fresh rat red cell in saline or plasma.     

Anatomical pathways from the white to the red pulp in the human spleen

In the human spleen we failed to find marginal zone bridging channels which in rats and mice are said to serve as return routes for lymphocytes from the white into the red pulp. In human spleens, using anticartilaginous antisera which distinctly visualized extracellular structures, in some parts we found the periarterial lymphocyte sheath to be closely attached to the red pulp, so that lymphocytes and other material could pass from the white pulp directly into the red pulp and vice versa. The strips of reticular fibres that seemed to bridge the marginal zone between the follicles and the red pulp proved to be components of reticular structures around the arteries, passing from the periarterial lymphocyte sheath into the follicles or from the follicles through the marginal zone into the red pulp.     

Comparative histochemical and electron microscopic studies of the sinus and venous walls of the human spleen with special reference to the sinus-venous connections

Sinus and venous walls of normal human spleens were studied with enzyme histochemical and electron microscopic methods. Particular attention was paid to the connections between sinuses and veins. Histochemically the sinus lining cells revealed a distinct naphthol-AS-acetate-esterase activity but no reaction for alkaline phosphatase. Venous endothelial cells were positive for the latter but negative for the former enzyme. In the sinus-venous junctional area there were no endothelial cells with reactivity for both enzymes. Electron microscopically both the sinus lining cells and the venous endothelial cells could be clearly characterized and therefore easily distinguished from one another on morphological grounds. There were no clear ultrastrural indications of transitional forms between sinus lining cells and venous endothelial cells in the sinus-venous area. According to these findings, sinus lining cells represent a specialized endothelium, but one with practically no morphological similarities to the venous endothelium.     

Stereometry of the red pulp on the human spleen. II) The normal spleen

The stereometric measurements obtained in three human normal spleens surgically removed for trauma have been submitted to statistical evaluation. On the basis of the original counts, some stereometric measures of the normal splenic red pulp have been determined, namely per cent volume of sinuses, per cent volume of cords, breadth of the cords, mean sectional area of sinuses and the volume of sinuses:volume of cords ratio. These data can constitute a series of parameters to which compare the measures of the pathologic spleens.     

Improved preparation of the integral membrane proteins of human red cells, with special reference to the glucose transporter

Human red cell membranes were isolated and partially stripped of peripheral proteins by gel filtration of hemolysates on a Sepharose CL-4B column at pH 8 connected in tandem to a Sepharose CL-6B column at pH 10.5. The eluted material was washed by centrifugations, once at pH 10.5 and twice at pH 12. In this way, water-soluble proteins and peripheral membrane proteins were thoroughly removed, and 0.2 g of integral membrane proteins could be prepared within 10 h from 0.2 litre of red cells. The exposure to high pH did not lower the D-glucose transport activity, and electrophoretically pure glucose transport protein could be isolated from this preparation. Gel filtration in sodium dodecyl sulfate separated the integral membrane components into four fractions, one of them containing 4.5-material; gel electrophoresis showed about 14 zones and two-dimensional electrophoresis resolved up to 100 mostly minor components, among which the glucose transporter focused around pH 7. However, purified glucose transporter focused around pH 8. Glucose and nucleoside transport proteins were co-purified in active form on DEAE-cellulose and a fraction isolated by adsorption to Mono Q was used for immunization of mice and production of monoclonal antibodies. One hybridoma produced antibodies that reacted with material in the 4.5-region, possibly the glucose transport protein, and not with band 3-material. Upon two-dimensional electrophoresis of integral membrane components that had been solubilized with octyl glucoside the immunoreactive and the silver-stained 4.5-material focused in a broad range from pH 6 to pH 9. A possible explanation for this heterogeneity might be interaction between the glucose and nucleoside transport proteins and negatively charged lipids.     

The tonicity-volume relations for human red cells subjected to the action of heat,with special reference to prolytic K loss

1. The volume-tonicity relations for human red cells exposed to a temperature of 48 degrees C. for 2 minutes remain the same as those for unheated human red cells. The heated systems show lysis in higher tonicities than the unheated systems do; this is probably largely due to fragmentation with its effect on the geometry of the situation, as suggested by Ham, Shen, Fleming and Castle. When the cells are heated to 48 degrees C. for longer times, the amount of fragmentation becomes considerable, but the volume-tonicity relation remains the same as before; the properties which are usually referred to as the osmotic properties of the red cell are accordingly not necessarily dependent on the integrity of the cell as a unit. 2. Heating to 52 degrees C. for 2 minutes profoundly modifies the volume-tonicity relation, very little swelling now occurring even in tonicities as low as 0.6. This is partly accounted for by the large K losses and K-Na exchanges which occur and which become greater as the tonicity is reduced and as the temperature is increased. Fragmentation and hemolysis also increase, the latter out of proportion to the expected effects of the former. Direct effects of heat on the cohesion of the red cell ultrastructure are probably involved.     

The red pulp of the human spleen. Structural basis of blood filtration

The paper summarizes the authors' findings indicative of an open blood circulation in the human spleen, particularly those obtained by immune and enzyme histo- and cyto-chemical methods, and by electron microscopy. In the red pulp the blood gets into the extravascular spaces of the pulp cords, where the individual blood components have to pass between numerous macrophages to reach the sinuses. The sinus wall is composed of elongated endothelial cells surrounded by waved annular or ring fibers of the basement membrane. In some areas annular fibers are joined by longitudinal fibers, giving rise to the filtration lattice, the fenestrated basement membrane. The sinus wall represents the last filter barrier which decises whether the blood elements get back into the blood or not. Extravasation of blood secures that all foreign as well as the altered own components, particularly cellular and particle ones naturally along with the normal constituents get from the circulating blood into extravascular spaces. In the next phase, however, the normal ones return into the circulation, whereas the abnormal components are removed from the extravascular tissue by means of the macrophagic and immune system of the spleen.     

Interdigitating cells in the white pulp of the human spleen

Summary Interdigitating cells are demonstrated as a special type of fixed cell in the periarteriolar lymphocytic sheaths of the human spleen. These cells show typical ultrastructural features as well as a characteristic enzyme histochemical pattern that distinguish them from other reticular cells in the splenic white pulp.     

Stereometry of the red pulp on the human spleen. I) Methodological remarks

The principal histometrical formulas to be applied to the analysis of human spleen's red pulp are illustrated. The discussion particularly concerns the formulas to evaluate the following measures per unit splenic volume:volume of the elements; surface area of sinus-splenic cord boundaries; mean breadth of cords; length of the sinus; mean sectional area of the sinus. Suggestions are given about the criteria to be followed in the proper assumption of the data which must be subjected to statistical estimation.     

The argentophil reticular cells of the mammalian spleen. II. The argentophil reticular cell arrangement inside the red pulp and its relationship with the endothelial lining cells of the splenic sinuses

The red pulp's argentophil reticular cell network of the spleen is composed by 3 types of fixed cells: 1. the primitive reticular cell, slightly argentophil; 2. the small reticular cell; 3. the larger reticular cell, strongly argentophil and phagocytic. This latter shows the classical morphological characteristics attributed to the reticular cells of the spleen. The large argentophil reticular cell may become free, constituting a 4th cell type, the free macrophage. A 5th reticular cell type is the dendritic cell found into the lymphatic follicles of the white pulp. The argentophil reticular cells of the red pulp assemble together to form the reticular cells' network, that occurs inside the red pulp cords. The primitive and the small reticular cell form the fundamental network on which the large cells are apposed. The reticular cells of this network maitain relationship with the arterial terminal vessels of the red pulp, being responsible by the ellipsoid structure. In those arteriolar segments without ellipsoid and in those mammalian species devoid of ellipsoid, the white pulp reticular cells, that surround the blood vessel as a part of the lymphoid periarteriolar sheath, mix with the red pulp's reticular cells and both can hardly be discriminated. The ellipsoids are formed by large argentophil cells arranged in concentrical layers around its lumen that sometimes appear devoid of endothelial lining cells. The red pulp's argentophil reticular cells, either the small or the large ones, contributed to the structure of the splenic sinuses' wall; its thin processes surround the sinus wall outside the endothelial lining cell as fibrillar structures that cross the back side of the lining cells. Two or more argentophil reticular cells send fibrillar processes to a single sinus. The perisinusal reticular cells may send a process between adjacent endothelial lining, cells that insinuate and attain the sinus lumen; this process becomes thick and eventually, the reticular cell enter the sinus lumen as a free macrophage. The argentophil reticular cells of the red pulp make connection between the capsule or the trabeculae and the reticular cell network. The endothelial lining cells of the splenic sinuses are not argentophil.     

Distribution of horseradish peroxidase (HRP)-anti-HRP immune complexes in mouse spleen with special reference to follicular dendritic cells

The distribution of immune complexes has been studied in mouse spleen stimulated to contain many germinal centers (GC's). Horseradish peroxidase (HRP)-anti-HRP complexes were used as an appropriately precise and sensitive model. We were primarily interested in the relative abilities of three cell types to interact with complexes: lymphocytes, macrophages, and follicular dendritic cells (FDC's). The latter are distinctive, nonendocytic, stellate cells located primarily at the transition of mantle and GC zones of 2 degrees lymphoid follicles (Chen, L. L., J. C. Adams, and R. M. Steinman, 1978, J. Cell Biol. 77:148). Binding of immune complexes to lymphocytes could not be visualized in situ. Macrophages avidly interiorized complexes into lysosomes, but did not retain them extracellularly. In contrast, FDC's could retain HRP-anti-HRP extracellularly under appropriate conditions, but did not endocytose them. Cytochemical reactivity accumulated progressively on FDC's 1--6 h after administration of complexes i.v., remained stable in amount and location for 1 day, and then was progressively lost over a 1- to 5-day period. Several variables in the association of complexes with macrophages and FDC's were pursued. Only 1 microgram of complexed HRP had to be administered to visualize binding to both cell types. Macrophages interiorized complexes formed in a wide range of HRP/anti-HRP ratios, while FDC's associated with complexes formed in HRP excess only. Quantitative studies with [125I]HRP-anti-HRP demonstrated that 20% of the splenic load of HRP associated with FDC's. Complexes formed with an F(ab')2 anti-HRP were distributed primarily in macrophages. When the levels of the third component of serum complement were depleted by prior treatment with cobra venom factor, uptake of complexes by macrophages was reduced some 50% whereas association with FDC's was abolished. The fact that antigen excess complexes are retained extracellularly strengthens the idea that they are immunogenic. Finally, the association of complexes with FDC's seems to retard the entry of antigen into the GC proper.     

Proteomics of chondrocytes with special reference to phosphorylation changes of proteins in stretched human chondrosarcoma cells

For proteomic analysis, cartilage molecular composition is a challenging mixture of highly glycosylated proteoglycans and triple-helical collagens, which constitute the major part of cartilage macromolecules. Selective separation of these molecules from the minor components is generally needed before mass spectrometry-based identification of lower-abundancy proteins is possible. The cell density of cartilage is also very low, therefore, cell cultures offer an easier approach to study cellular responses of chondrocytic cells, e.g., to mechanical stimuli. In this study, we investigated the phosphorylation events in human chondrosarcoma cells during cellular stretching. Human chondrosarcoma cells were stretched to 8% strain at a frequency of 1 Hz. One set of experiments included cellular stretching which lasted 2 hours, and the other one included experiments of 2 hours daily treatment for up to 3 days. Two-dimensional polyacrylamide gel electrophoresis combined with chromatographic phosphoprotein pre-enrichment and electrospray ionization mass spectrometry-based protein identification was used to reveal changes of phosphoproteins in cells exposed to cyclic stretching. We discovered that 2 hours cyclic stretching increased the phosphorylation of moesin, elongation factor eEF1D and leprecan, while the phosphorylation of elongation factor eEF1B decreased after cellular stretching. Western blot analyses with phospho-specific antibodies suggested that stretching induces phosphorylation of ERK of the MAP kinase pathway, but did not induce phosphorylation of phosphatidylinositol 3-kinase. In conclusion, the proteomic approach revealed that cellular stretching induced specific phosphorylation changes in chondrosarcoma cells.