Hemolymph cells and the platelet-like structures of the land slug, Incilaria bilineata (Gastropoda: Pulmonata)

3 morphologically distinct hemolymph cell-types, Types I, II and III, were found by light and electron microscopy of hemolymph from the land slug, Incilaria bilineata. In addition, vast numbers of the platelet-like structures were present in the collected hemolymph. Type I cells were macrophage-like, Type II cells were lymphocyte-like and Type III cells were fibroblast-like. Among these hemolymph cells, only Type I cells could recognize foreign materials and phagocytose them. Type I cells, whether from circulating hemolymph or from the heart-kidney region (regarded as a hemopoietic organ), could be maintained for only 4 d in cultivation because the cells gradually divided within 3 or 4 d. Thus, the platelet-like structures appear to be derived from Type I cells, which readily fragmentize and function by clamping onto foreign materials.     

The uptake and long-term storage of India ink particles and latex beads by fibroblasts in the dermis and subcutis of mice, with special regard to the non-inflammatory defense reaction by fibroblasts

The fate of India ink particles and polystyrene latex beads injected into the dermis and subcutis of the skin of the auricle and back in mice was observed with the naked eye, light microscopy and electron microscopy. The tattoo patterns made by injected ink particles remained essentially unchanged for life as observed with the naked eye. India ink particles and latex beads were endocytosed by fibroblasts as well as macrophages in the dermis and subcutis. Numerous ink particles or small latex beads (0.22 micron in diameter) were packed into vacuoles 0.1-10.0 micron in diameter which occupied a large volume of the cytoplasm of the cell body and processes of fibroblasts, whereas numerous particles and larger beads (0.22 and 2.0 micron) were taken up into the cell body of macrophages in the vicinity. Most fibroblasts, characterized by long cell processes and well developed rough endoplasmic reticulum, are easily distinguished from macrophages, the latter being round or oval in shape, and having many lysosomes and numerous irregularly shaped microvillous projections. It is believed that fibroblasts taking up and storing the ink particles or latex beads move poorly and are almost fixed in the connective tissue: the tattoos therefore do not change markedly. It is emphasized that the uptake and long-term storage of ink particles and latex beads by the dermal and subcutaneous fibroblasts represent a specific non-inflammatory defense mechanism that protects the living body, without immune reactions, against injuries and invasions by non-toxic foreign agencies. The histiocyte, a term proposed by KIYONO (1914) for a fixed macrophage on the basis of his studies using vital dye staining, is considered to include, in addition to true macrophages, fibroblasts showing endocytotic activities for small foreign bodies such as acid dyes, ink particles, and latex beads.     

Endocytotic activity of the free floating cells and epithelial cells in the endolymphatic sac: an electron microscopic study

The fine structure and its functional properties of both free floating cells and epithelial cells in the endolymphatic sac after injections of India ink particles or polystyrene latex beads, 0.24 micron in diameter, into the endolymphatic space, were studied using light and electron microscopy. Twenty-four to 48 hours after injections, these foreign materials had accumulated in the lumen of the endolymphatic sac and a large number of them were ingested into free floating cells, most of which appear to be macrophages. Granular leucocytes taking up the foreign materials into the cytoplasm were also recognized in the sac lumen. A few of these leucocytes passed through the epithelium and migrated to the subepithelial connective tissue, while the others were degenerated and phagocytosed by the free floating cells in the lumen of the sac. In addition to the free floating cells, ink particles, latex beads, and degenerated leucocytes were endocytosed into some of the epithelial cells. As we reported previously, the epithelial cells were clearly classified into two types; type-1 epithelial cells (cytoorganelle-rich cells) and type-2 epithelial cells (filament-rich cells). The foreign materials and degenerated cells were taken up mainly into the type-1 epithelial cells, while the type-2 epithelial cells did not show so marked endocytotic activity in comparison with the type-1 epithelial cells. Thus, it becomes clear that the endolymphatic sac plays an important role in the endocytotic activity for foreign materials and waste products, and both the free floating cells and the type-1 epithelial cells of the sac have strong endocytotic activity.     

The ultrastructure of hemolymph cells of the land slug, Incilaria fruhstorferi Collinge (Gastropoda: Pulmonata)

3 morphologically distinct hemolymph cell-types, Type I, II and III, were found in the hemolymph of the land slug, large Japanese native slug, Incilaria fruhstorferi Collinge. Type I cell, measuring approximately 35 microns in diameter, had extensive pseudopodia with supporting ribs and a kidney-shaped or lobulated nucleus. Sometimes it possessed long filopodia (approximately 50 microns) with branched fibers. It contained residual bodies, multivesicular bodies, Golgi apparatus, free ribosomes and glycogen-like deposits. Type II cell, approximately 5 microns in diameter, did not form large pseudopodia, had a higher nucleus to cytoplasm ratio. It contained scattered free ribosomes surrounding the round nucleus. Type III cell, approximately 75 X 15 microns, was a fibroblast-like cell. It contained microfibrils at the rim of perinuclear cytoplasm and possessed collagen-like fibers outside of the cytoplasmic membrane. Numerous plate-like structures, which had not been reported yet, were usually observed. They, measuring 1 to 5 microns in diameter, were oval, but when in contact with glass, they possessed long and fine fibers. They lacked nucleus.     

Exosomal-like vesicles are present in human blood plasma

Exosomes are small membrane vesicles (50-90 nm in diameter) secreted by most hematopoietic cells. We provide here the first evidence for the presence of exosomes in vivo, in the blood. Plasma samples of all healthy donors tested (n = 15) contain vesicles that are similar in shape, size and density to the previously described exosomes. They were clearly identified by electron microscopy after isolation by differential ultracentrifugation or immunoisolation with CD63-coated latex beads. We performed their biochemical characterization by western blot analysis and by flow cytometry after vesicle adsorption onto latex beads using a panel of mAbs. We observed that these plasma-derived vesicles contain tetraspanin molecules such as CD63, CD9, CD81 as well as class I and class II MHC molecules and Lamp-2 (i.e. proteins that are known to be enriched in exosomes). In addition, these vesicles float on sucrose gradient at a density similar to exosomes. Our results demonstrate that blood is a physiological fluid for exosome circulation in the body, suggesting their role in cell-cell or organ-organ communications as carriers for molecules that need to reach distant cell targets.     

Resident sinusoidal macrophages in the liver of the brown bullhead (Ictalurus nebulosus): an ultrastructural, functional and cytochemical study

Ultrastructural, functional, and cytochemical characteristics of resident sinusoidal macrophages (RSM) in brown bullhead (Ictalurus nebulosus) liver were examined. Following perfusion fixation of the hepatic vascular bed, light micrographs revealed RSM that possessed multiple elongate cytoplasmic processes and frequently contained erythrocytes in various stages of degradation. Following brief perfusion fixation, light microscope examination of vibratome sections of bullhead liver reacted for peroxidase revealed intensely positive RSM. By transmission electron microscopy, peroxidase activity was localized to the nuclear envelope and cytoplasmic granules of RSM and in endothelial and perisinusoidal fat-storing cells. In cryostat sections of fresh-frozen liver, glucose-6-phosphate dehydrogenase (G-6-PDH) was uniformly distributed over hepatocytes, whereas intensely positive punctate staining for G-6-PDH was localized over RSM. To test for phagocytosis by RSM, latex beads (0.81 micron) were injected into a tributary of the hepatic portal vein 2 min prior to perfusion fixation. Latex beads appeared either singly or in dense aggregates within RSM. Ultrastructurally, RSM were characterized by an irregularly shaped, eccentrically located nucleus, electron-dense vacuoles, small patches of granular endoplasmic reticulum, a well-developed Golgi apparatus, elongated mitochondria, desmosomes or desmosome-like densities that served as a source of attachment to endothelial cells, and a centriole with radiating microtubules. Invaginations of the plasma membrane (vermiform processes) characteristic of mammalian Kupffer cells were not observed in bullhead RSM. The results indicated a resident cell population of sinusoidal macrophages in the bullhead liver with properties that partially resembled mammalian Kupffer cells. These results are important for the identification of the normal resident cells in the bullhead liver.     

Generation of nanovesicles with sliced cellular membrane fragments for exogenous material delivery

We propose a microfluidic system that generates nanovesicles (NVs) by slicing living cell membrane with microfabricated 500 nm-thick silicon nitride (Si_xN_y) blades. Living cells were sliced by the blades while flowing through microchannels lined with the blades. Plasma membrane fragments sliced from the cells self-assembled into spherical NVs of ∼100–300 nm in diameter. During self-assembly, the plasma membrane fragments enveloped exogenous materials (here, polystyrene latex beads) from the buffer solution. About 30% of beads were encapsulated in NVs, and the generated NVs delivered the encapsulated beads across the plasma membrane of recipient cells, but bare beads could not penetrate the plasma membrane of recipient cells. This result implicates that the NVs generated using the method in this study can encapsulate and deliver exogenous materials to recipient cells, whereas exosomes secreted by cells can deliver only endogenous cellular materials.     

Evaluation of flow cytometry and fluorescence microscopy for the estimation of bovine mononuclear phagocytes

Bovine blood mononuclear cells were isolated by density gradient centrifugation on Ficoll-Paque. Phagocytic mononuclear cells were characterized functionally by ingestion of fluorescent latex beads. After incubation with beads the cells were treated with Triton X-100 and propidium iodide (PI) to stain DNA. Cells were analyzed with a FACS-III instrument connected to a Nuclear Data-6660 multiparameter computer system. The computer was used to evaluate the 2 parameter histograms in order to enumerate the percentage of cells with different numbers of associated beads. With this system we also obtained information about cell concentration and number of beads per cell. Results from flow cytometry and manual counting by fluorescence microscopy were compared and good correlation (r = 0.91) was obtained. During the first hours of incubation latex beads adhered to cell surfaces as demonstrated by FCM histograms and fluorescence microscopy. Blood mononuclear phagocytes have to be incubated for several hours before significant phagocytotic activity can be detected.     

Altered membrane fatty acids of cultured human retinal pigment epithelium persistently infected with rubella virus may affect secondary cellular function

Summary Persistent infection with rubella virus (RV) can alter secondary functions of host cells. Previously we had documented defective phagocytosis of latex beads by cultured human retinal pigment epithelial cells (RPE), persistently infected with M-33 RV (RPE/RV). Here, examining possible mechanisms for altered function, we reported significant differences between the total esterified fatty acids (FA) of RPE and RPE/RV membranes, measured by gas liquid chromatography. RPE/RV contained an increased proportion of saturated FA, particularly palmitic acid, with a presence of unusual chromatographic FA peaks co-eluting with odd-numbered long-chain carbon atom FA not normally found in human cells. Apical membrane microvilli, structures essential to phagocytic activity of RPE and RPE/RV, observed by scanning and transmission electron microscopy, were similar in number and appearance between uninfected RPE and RPE/RV cells before and after latex bead addition. However, RPE/RV microvilli, possibly reflecting altered membrane FA composition, engaged latex beads less effectively than uninfected RPE microvilli. In addition, microvilli remained abnormally distributed on RPE/RV cell surfaces at 48 h after latex addition. Thus, RV persistent infection may affect the cellular membrane fluidity and functional activity of human cells with increased saturated FA proportions and altered FA components of membrane phospholipids. These changes may participate in the defective phagocytosis of RPE/RV.     

Phagocytosis of latex beads is defective in cultured human retinal pigment epithelial cells with persistent rubella virus infection.

Phagocytosis, a secondary function of retinal pigment epithelial (RPE) cells essential to sight, was significantly decreased, when measured with latex beads, during persistent rubella virus (RV) infection of human cultured RPE cells. A target for RV in vivo, RPE cells infected with RV (RPE/RV) ingested fewer fluorescent microspheres (26%) than did uninfected RPE cells (68%) (P < 0.001), as measured by flow cytometry. In RPE/RV cells, with characteristic RPE monolayer appearance and normal growth during subculturing over 6 months, persistent RV infection was shown by specific RV antigen immunofluorescence, by the presence of the RV genome in RPE/RV cell messenger RNA, and by recovery of cell-free RV after cocultivation with Vero cells. The adhesion of latex beads to apical cell surfaces of RPE/RV and uninfected RPE cells appeared similar, as imaged by scanning electron microscopy. Cytoskeletal actin, a component of phagocytosis in RPE, appeared altered in 60 to 75% of RPE/RV cells by antiactin immunofluorescence staining, as previously described in other RV-infected cells, but its role in the disturbed phagocytosis of latex beads was not determined. Persistently RV-infected human RPE is an additional example of RV-associated secondary cellular dysfunction in the absence of cytopathic effects.     

Three-dimensional growth of human hepatoma C3A cells within alginate beads for fluidized bioartificial liver

OBJECTIVES: Alginate beads allow cultivation of cells in a 3-dimensional environment. The aim of our study was to assess the influence of a 3-dimensional culture in alginate microbeads, on hepatic cell metabolism. METHODS: We used 2 types of alginate: low viscosity (LV) and medium viscosity (MV). The hepatic cell line C3A was encapsulated in alginate beads. Cells were cultured for 2 weeks. Using scanning electron microscopy, the morphology of 3D structures and the surfaces of cells were analyzed. Fluidized bed bioartificial liver experiments were performed 24 hours, 7, and 14 days after bead formation. RESULTS: Two different cell growth types in alginate beads were observed: channel-like structures and spherical aggregates characteristic of LV and MV alginate, respectively. A significant increase in albumin synthesis was observed in long-term culture. Formation of characteristic hepatic cell microvilli on cell surfaces was observed under scanning electron microscopy for both types of alginate. Prolonged static cultivation of C3A cells within the alginate beads in both types of alginates caused significant increases in albumin production in the fluidized bioreactor. CONCLUSIONS: Cultivation of the hepatic C3A cells within the alginate microbeads significantly improved bioreactor effectiveness in albumin production. The presence of extensions of cell membranes on the surface of hepatoma cells in 3-dimensional culture within the alginate beads indicated formation of microvilli-like structures characteristic of normal hepatocytes.     

Alteration of the structure and function of guinea pig peritoneal macrophages by a soybean oil emulsion.

Studies in humans who have received Intralipid (IL) have demonstrated the presence of a fat pigment and fat droplets in reticuloendothelial phagocytic cells. Clinical data and in vitro studies suggest that these cells do not function normally. We have studied the effect of IL on the morphology and function of guinea pig peritoneal macrophages in vitro. Starch-induced macrophages were exposed to IL for up to 48 hours. Ingestion of increasing amounts of IL over the 48-hour period was confirmed by transmission electron microscopy and by oil red O stain. The uptake of the IL was associated with marked morphologic changes characterized by a decreased ability of the cells to spread and by a decrease in the number and degree of complexity of the membrane ruffles. The ingestion of IL also resulted in decreased capacity of the cells to associate with latex beads (5.7 mu in diameter) or Candida albicans and decreased capacity to adhere to and ingest sheep erythrocytes coated with IgG. After ingestion of latex beads 0.46 mu in diameter, which are similar in size to IL particles, macrophages had normal morphology and function, indicating that neither the morphologic nor functional abnormalities were due to a nonspecific effect of ingestion of small particles. Alterations of human reticuloendothelial macrophage function similar to the effects observed here could compromise host defense against infection.     

Bovine circumvallate taste buds: taste cell structure and immunoreactivity to alpha-gustducin

The taste buds of bovine circumvallate papillae were investigated under light and electron microscopy both by histological and immunohistochemical methods. Taste buds existed in the inner epithelium of the trench of the papillae. Under electron microscopy, two types of taste cells, type I and type II, could be classified according to the existence of dense-cored vesicles and cytoplasmic density. Type I had electron-lucent cytoplasm and possessed many electron-dense cored vesicles in the apical cytoplasm. It was considered that the electron-dense materials of the vesicles were released and constituted the pore substance. This type of cell possessed long and thick apical processes in the taste pore. Type II had denser electron cytoplasm compared with that of type I and possessed many electron-lucent vesicles in the apical cytoplasm. This type of cell possessed microvilli in the taste pore. To know the immunoreactivity to alpha-gustducin in bovine circumvallate taste buds, we used the immunoblotting method and the immunohistochemical method. The alpha-gustducin reaction band at 40 kDa was displayed in the specimen of Western blots. The immunohistochemical property of the antiserum to alpha-gustducin was investigated by using the avidin-biotin complex (ABC) method and the 1.4-nm gold and silver enhancement methods. A subset of taste cells showed the immunoreactivity under light microscopy. The electron microscopic specimens with the 1.4-nm gold and silver enhancement method revealed that only type II cells exhibited the alpha-gustducin immunoreactivity.     

Cilia of neonatal articular chondrocytes: incidence and morphology

Cilia in neonatal canine articular chondrocytes were studied using morphometric techniques and transmission electron microscopy. The cilia in chondrocytes were morphologically similar to cilia in a variety of other cell types. A chondrocytic cilium consisted of a basal body and a ciliary shaft. The cylindrical basal body was 0.21 micron (S.D. = 0.01 micron) in diameter, 0.50 micron (S.D. = 0.03 micron) in length and contained nine microtubular triplets. The ciliary shaft was 0.196 micron (S.D. = 0.02 micron) in diameter and 1.76 micron (S.D. = 0.80 micron) in length. The number of microbtubular doublets in the ciliary shaft varied depending on where along the length of the shaft the section was taken. This study demonstrates that on the average the frequency of cilia in neonatal articular chondrocytes, as estimated sterologically, was about one cilium per cell.     

Lymphokine-stimulated macrophage phagocytosis of fluorescent microspheres: a rapid new assay

A highly sensitive and rapid in vitro macrophage phagocytosis assay is described for screening of lymphokine preparations with macrophage activating properties. Non-induced mouse peritoneal macrophages were incubated on glass slides for 60 min in the presence of fluorescent 2 micron latex beads and partially purified lymphokine fractions or media control. Lymphokine samples were prepared from culture supernatants of the B lymphoblastoid cell line RPMI 1788 by high performance liquid chromatography of a soluble trichloroacetic acid extract of concentrated culture supernatant. Phagocytosis was measured by direct counts of the percent phagocytic cells and the number of intracellular beads per 100 cells as seen by fluorescence microscopy. Phagocytic uptake in the presence of as little as 0.1 ng of active lymphokine could readily be determined qualitatively by correlation plots and quantitatively by appropriate statistical programs for data processing by computer. This technique provides a rapid, reproducible, screening assay for biological activity of macrophage activating lymphokines and other compounds affecting macrophage phagocytosis.     

Type I and IV collagen and fibrinogen binding to Aeromonas species isolated from various infections

Collagen binding is a common property of strains of Aeromonas species. However, agglutination of latex beads coated with types I and IV collagen and fibrinogen with Aeromonas cells varied among strains of Aeromonas species and their source of isolation. Culture media and growth conditions greatly influenced expression of Aeromonas cell surface receptors to bind collagen (types I and IV) and fibrinogen immobilized on the latex particles as suggested by the particle agglutination assay (PAA). Aeromonas cells aggregated with the differentially coated latex beads in a specified manner. Furthermore, the PAA method was found to be rapid, easy to perform and sensitive for routine screening of a large number of strains for serum and connective-tissue protein cell surface receptors.     

Deficient Kupffer cell phagocytosis and lysosomal enzymes in the endotoxin-low-responsive C3H/HeJ mouse

Various substances, including lysosomal enzymes, are produced by Kupffer cells and other macrophages; their release has been implicated in the toxic response to endotoxins. C3H/HeJ mice exhibit little or no response to doses of endotoxin that are lethal in syngeneic C3HeB/FeJ mice. To explore the nature of this deficient response, the Kupffer cells of these mice were studied using in vivo microscopic as well as histochemical and electron microscopical methods. In vivo, the rate of phagocytosis of single 0.8 micron latex particles was measured in individual Kupffer cells as was the number of phagocytic cells per microscopic field. Frozen sections of livers were stained for a variety of lysosomal enzymes and liver specimens also were processed for electron microscopy. In comparison to the endotoxin-sensitive C3HeB/FeJ mice, the livers of the C3H/HeJ mice contained 60% fewer Kupffer cells that phagocytosed latex. However, the rate of phagocytosis by these cells was not statistically different and ranged from 19-26 sec. The volume density of acid-phosphatase-positive Kupffer cells was 40% less in the C3H/HeJ mice. Similar differences were observed with other lysosomal enzymes including cathepsins B and H and dipeptidyl peptidases I and II. However, light and electron microscopy revealed a relatively normal number of Kupffer cells in livers stained for peroxidase, a nonlysosomal enzyme. The results suggest that the insensitivity of C3H/HeJ mice to endotoxin may be related in part to a lysosomal enzyme deficiency and a paucity of phagocytic Kupffer cells in these animals.     

Ontogeny of macrophage function. I. Phagocytic activity and A-cell activity of newborn and adult mouse peritoneal macrophages.

Phagocytic activity and immune-participating A-cell activity of newborn and adult mouse macrophages were investigated under in vitro conditions. Thioglycollate medium-stimulated newborn (SNB) or adult (SA), and nonstimulated adult (NA) peritoneal macrophages were used. Immune complexes of sheep erythrocytes (SRBC), aldehyde-fixed SRBC, and latex beads were employed in phagocytosis tests. A-cell activity was estimated as the capacity to support primary or secondary responses of macrophage-deprived adult spleen cells to SRBC. Results obtained were: 1) phagocytic activity of NA macrophages was highest and that of SNB macrophages was higher than SA macrophages, 2) no difference was observed in A-cell activity for secondary response among SNB, SA, and NA macrophages, and 3) SNB macrophages were lacking in A-cell activity for primary response. These results suggest that the substantial A-cell function should be evaluated in primary responses, and that the A-cell and pahgocytic functions do not necessarily accompany each other.     

Scanning and transmission electron microscope observations of the terminal segment of the cat seminiferous tubule: epithelial phagocytosis of spermatozoa and latex beads

The terminal segment of the seminiferous tubule in the testis of the adult domestic cat was investigated using scanning and transmission electron microscopy. The terminal segment gradually tapers towards the tubulus rectus, while transforming into tall columnar supporting cells termed modified Sertoli cells which protrude into the lumen of the tubulus rectus in the form of a plug. There is no distinct central lumen in the plug, rather, it is a narrow slit-like cleft surrounded by the modified Sertoli cells. The modified Sertoli cells of the plug as well as the cuboidal cells lining the tubulus rectus revealed an active phagocytosis of spermatozoa and even injected inert latex beads. These epithelial cells are thought to act as a protective filtration line for foreign bodies including unwanted spermatozoa.     

A light and electron microscopic study on pulpal nerve fibers in the lower incisor of the mouse.

Light and electron microscopic studies were made on pulpal nerve fibers in mouse lower incisors, typical continuously growing teeth. Serial sections, from the apex of the odontogenic sheath to the incisal edge of the apical foramen, were examined by light microscopy to identify myelinated fibers passing through the apical foramen. The fine structure of the pulpal nerves was examined by electron microscopy at three sites: 1) the level at the incisal edge of the apical foramen; 2) a level 5 mm incisal from the apex of the odontogenic sheath; and 3) the level where the incisor comes out of the alveolar bone. No myelinated fibers were found passing through the apical foramen; they were also lacking at the three levels of the pulp. At level 2, unmyelinated axons were seen in close contact with smooth muscle fibers of arterioles. At level 3, nerve fibers were difficult to distinguish from processes of fibroblasts and odontoblasts. Degenerating axons were present in Schwann cells, and fine unmyelinated axons running through the odontoblast cell layer were seen. Various types of unmyelinated axons were observed in the apical region (level 1). These axons were classified into 6 types on the basis of their fine structures: Type I, bundles of unmyelinated axons completely or partly ensheathed by Schwann cell cytoplasm (mature type); Type II, bundles of unmyelinated axons in a space formed by a Schwann cell membrane (regenerating type); Type III, bundles of unmyelinated axons ensheathed not by a Schwann cell, but merely by a basal lamina (regenerating type); Type IV, single axons in direct contact with the basal lamina (regenerating or terminal type); Type V, naked, electron-dense axons with many vesicles and mitochondria (growth cone-like type); and Type VI, electron opaque axons, due to loss of axonal organellae (degenerating type). The significance of these structures is discussed in relation to the continuous growth of the rodent incisor.