A scanning electron microscope study of mouse peritoneal mesothelium.

As seen in the scanning electron microscope, peritoneal mesothelial cells of the mouse diaphragm, anterior abdominal wall and intestinal serosa carry numerous microvilli. These microvilli are absent over certain areas of the cell surface and are sometimes, interlocked in meshwork patterns or coronal formation. The apical cell membranes of the mesothelium at the base of the microvilli, are invaginated by many plasmalemmal vesicles and vacuoles and carry a number of protruding spherical structures. Deep circular craters, giving the impression of stomata, are also visible.     

Phagocytic uptake of latex beads by rat peritoneal macrophages: Comparison of morphological and radiochemical assay methods

Contrary to previous reports, commercially available 1000-nm latex beads were found to be labelable with¹²⁵I, yielding a product that retained its radiolabel on storage at 4°C and when incubated in tissue-culture media. This finding permitted a radiochemical method to measure phagocytic uptake of latex particles by rat peritoneal macrophages cultured in vitro, and a direct comparison with the established method of particle counting by light microscopy. The two methods yielded closely similar data, demonstrating that the (much more convenient) radiochemical method for quantitating phagocytic uptake is both feasible and reliable. The kinetics of phagocytic uptake of the latex particles and the effect of low temperature and metabolic inhibitors (sodium fluoride and 2,4-dinitrophenol) are described. Ongoing phagocytosis did not alter the rate of fluid-phase pinocytosis by macrophages.     

New sensitive light microscopical detection of colloidal gold on ultrathin sections by reflection contrast microscopy. Combination of reflection contrast and electron microscopy in post-embedding immunogold histochemistry.

One simple post-embedding method for combined light- and electron microscopy is presented. Different types of antigens in normal rat and mouse kidneys as well as in tissues from cases of experimental induced nephritis were stained after Lowicryl K4M embedding by an immunogold (silver) method. The (silver-enhanced) gold particles were visualized by light microscopy, e.g. bright-field (BFM)- and reflection contrast (RCM) microscopy, as well as by electron microscopy. The potentials of RCM visualization in this field were investigated, resulting in the successful detection of colloidal gold (15 nm) particles, or silver enhanced gold particles, on ultrathin sections. Furthermore, an increased detection sensitivity of RCM compared with BFM together with an increase in the sensitivity of the immunostaining by RCM visualization was found. The different ways to use RCM, alone or in combination with bright-field- or phase contrast microscopy for visualization of plastic sections varying in thickness, type of plastic and staining, are discussed.     

The coating of mouse myocardial cells. A cytochemical electron microscopical study.

The coating of mouse myocardial cells has been investigated with a variety of cytochemical methods. The coating of the surface membrane gives a positive reaction with ruthenium red, colloidal thorium, phosphotungstic acid (PTA) at low pH, silver methenamine after periodic oxidation (PA-silver technique) and with silver proteinate after periodic oxidation and thiocarbohydrazide treatment (PA-TCH-silver technique). The coating of the T system gives almost similar results. The nexuses do not react with PTA nor with the PA-silver and PA-TCH-silver techniques, but they are strongly stained with ruthenium red which reveals periodic structures in their gaps. The specificities of the colloidal thorium technique and PAT staining have been tested by chemical treatments (methylation, acetylation, saponification), enzymatic digestions (pronase, trypsin, hyaluronidase, neuraminidase) and carbohydrate extractions (with 0.1 N NaOH and 0.05 M H2SO4). These cytochemical data indicate, considering the specificity of the reactions, that the coating of the membrane surface and the T system contains polyanionic groups. A part of them, at least, would belong to a carbohydrate-containing material (glycoproteins), whereas at the level of nexuses the sugar residues would probably be absent.     

Cuticular structures of the priapulid Halicryptus spinulosus: A scanning electron microscopical study

Summary The surface anatomy and the structures lining the pharynx of Halicryptus spinulosus were examined by scanning electron microscopy (SEM). The structures were compared and contrasted with those reported for other priapulids, particularly those features previously studied with SEM. Buccal papillae and pharyngeal teeth of two types were described. Surface structures observed with SEM were: scalids, abdominal setae, anal papillae, posterior warts and ring papillae. The latter three structures are unique among described priapulids. The anal papillae are composed of several rounded, perhaps pedunculate, structures; the posterior warts are composed of mitriform structures in close association with columnar structures. Both are located in separate depressions in the posterior integument. The ring papillae occur on the annuli close to the posterior end. Halicryptus spinulosus was previously thought to lack these structures.     

黄鳝咽部表面形态结构扫描电镜研究

用扫描电子显微镜对黄鳝咽部表面亚微形态结构作了详细的观察研究.黄鳝咽部表面具有许多纵行的褶,细胞间具有深沟、深洞和不规则的颗粒等不同结构.咽部上皮细胞表而结构与其它鱼类鳃弓、鳃丝、鳃耙上皮细胞相似,都具有隆嵴、短纹和点纹.这些表面形态结构与黄鳝咽部呼吸机理密切相关.     

Utilization of gammalinolenic acid by mouse peritoneal macrophages.

To delineate the metabolism of gammalinolenic acid (18:3(n-6] by macrophages, primary cultures of resident mouse peritoneal macrophages were incubated with [14C]18:3(n-6). At 3, 6 or 20 h, the majority (greater than 85%) of the radiolabel was recovered in cell phospholipids. With increasing time of incubation, a relative reduction of 14C in glycerophosphocholine (ChoGpl, 58.1% to 46.2%) was noted. This was offset by a corresponding increase in glycerophosphoethanolamine (EtnGpl) labeling (from 8.8% to 18.9%). There was also a time-dependent redistribution of 14C from diacyl to ether-containing phospholipid subclasses in ChoGpl and EtnGpl. Analysis of cell extracts by reverse-phae HPLC following transmethylation demonstrated that 18:3(n-6) was extensively elongated (greater than 80%) to dihomogammalinolenic acid (20:3(n-6] by 3 h. The major radiolabeled phospholipid molecular species in the diacyl (PtdCho) and alkylacylglycerophosphocholine (PakCho) subclasses was 16:0-20:3(n-6). In contrast, diacyl (PtdEtn) and alkenylacylglycerophosphoethanolamine (PlsEtn) subclasses contained primarily [14C]18:0-20:3(n-6) and 16:0-20:3(n-6), respectively. Macrophages prelabeled with [14C]18:3(n-6) for 20 h and stimulated with calcium ionophore A23187 or zymosan synthesized [14C]prostaglandin E1 (PGE1). These data demonstrate that macrophages possess an active long chain polyunsaturated fatty acid elongase capable of converting 18:3(n-6) to 20:3(n-6) which can, upon stimulation, be converted to PGE1.     

A prothrombinase complex of mouse peritoneal macrophages

Addition of prothrombin to mouse peritoneal macrophages in vitro resulted in the formation of a thrombin-like enzyme, as demonstrated by use of the luminogenic peptide substrate S-2621. The prothrombinase activity was sedimented by high-speed centrifugation following homogenization of the cells and was abolished by treatment of the cells with the nonionic detergent Triton X-100 at 0.02% concentration. Moreover, the activity was drastically reduced by maintaining cultures in the presence of warfarin and, presumably due to competitive substrate inhibition, by adding S-2222, a chromogenic peptide substrate for Factor Xa. These findings suggest that prothrombin cleavage is catalyzed by Factor Xa at the macrophage surface. The generated thrombin was inhibited by antithrombin, and this reaction was accelerated by heparin with high affinity for antithrombin but not by the corresponding oligosaccharides composed of 8-14 monosaccharide units. Such oligosaccharides which are capable of accelerating the inactivation of Factor Xa by antithrombin, inhibited thrombin formation from prothrombin in the macrophage cultures, presumably by promoting inactivation by antithrombin of Factor Xa in a prothrombinase complex. Activation of the macrophage coagulation system, as proposed to occur in certain inflammatory conditions, thus may be modulated at various levels by heparin, or heparin oligosaccharides, released from mast cells.     

Uptake and degradation of mast-cell granules by mouse peritoneal macrophages.

35S-labelled mast-cell granules isolated from mouse mastocytomas were added to mouse macrophages in vitro. The granules were avidly phagocytosed, and subsequently the radioactivity was released to the medium as inorganic [35S]sulphate. After pulse-labelling, a total of about 80% of the cell-associated radioactivity was thus released in the course of 24 h, indicating an extensive breakdown of the sulphated polysaccharides, mainly heparin, present in the granules. The uptake of the mast-cell granules caused pronounced, but reversible, spreading of the macrophages.     

The morphology of the denuded epidermal basal cell layer of the hairless mouse after different preparation methods. A scanning and transmission electron microscopical study

The denuded basal cell layer of the hairless mouse epidermis is described in the present scanning (SEM) and transmission electron microscopical (TEM) study. The suprabasal layers were removed mechanically after trypsinization or by extracellular calcium depletion. Trypsinization before removal of the suprabasal cells caused the basal cells to shrink. Characteristic surface plication and hemi-desmosomal attachment to the basement membrane were generally preserved. SEM revealed partly maintained intercellular bridging, whereas by TEM such contacts were absent because half desmosomes were internalized. Total calcium depletion induced more serious damage to the basal cell surface, which was smooth with apparent perforations. However, cell bridges, and occasional desmosomes were present. The cell interior demonstrated important cellular injury. If the calcium deprived explants were allowed to recover in calcium-containing medium, the cells acquired an activated regenerative morphology, without junctions, similar to that observed in wound healing. Epidermal non-keratinocytes were seen only after trypsinization. Control experiments revealed that they adapted poorly to organ culture conditions. By TEM, we observed several interesting aspects of the differences, between dark and clear basal keratinocytes. This was unexpected because fixation studies had shown, that with the present fixation method, typical dark and clear cells do not occur in untreated epidermis. We believe that membrane injury through mechanical stripping of partly adhering epidermal layers induced clear cells, whereby the neighboring cells appeared darker. This provides additional evidence as to the origin of the two sub-populations, dark and clear basal cells. The clear cells may be injured cells, caused by cell damage, and not by processes of cellular differentiation. The results of the present investigation supports the view that basal keratinocytes have a polygonal shape with numerous free surface extensions and they are anchored to the basement membrane with foot pads. Our study also shows that SEM of the epidermal basal layer might be feasible. Various artifacts, however, must be considered, depending on the denudation method used. We prefer trypsinization to calcium depletion because it is less time-consuming and results in a cell morphology which in TEM is comparable to that of basal cells in untreated whole epidermis. Extra-cellular calcium depletion, however, might be useful as a method to prepare single cell suspensions for flow cytometry. Restoration of a normal calcium concentration after stripping, provides an opportunity to mimic wound healing in situ, as an alternative t     

The mouse vomeronasal glands: a light and electron microscopical study

The glands of adult mouse vomeronasal organ (VNO) were studiedwith light- and electro-microscopical techniques. The vomeronasalglands (VN-Gs) consist of several individual glandular complexesdistributed along the long axis of the VNO. The secretory productsreleased from VN-G cells enter into the lumen of the VNO inthe region of transition between the neuroepithelium and thereceptor-free epithelium. The acini show the typical morphologicalfeatures of serous glands. The secretory cells of these aciniare characterized by a round to oval nucleus and a well-developed,rough endoplasmic reticulum, both preferentially located inthe basal part of the cell. The supranuclear region is occupiedby the Golgi apparatus and secretory granules varying in sizeand electron density. They accumulate towards the apical partof the cell. Secretory cells are connected by tight junctions,desmosomes and membrane interdigitations, moreover, they arealso coupled by gap junctions. Axonal terminals containing clearvesicles and dense-cored vesicles are frequently seen betweenthe secretory cells. Secretory cells are directly related tothe thin basal lamina of the acinus; myoepithelial cells arenot present. In the lamina propria, numerous smooth muscle cells,blood vessels and nerve bundles containing both myelinated andunmyelinated axons can be observed. An automatic regulationof the activity of the VN-Gs is discussed in relation to thevomeronasal pump.     

A stereological ultrastructural study of stimulated peritoneal macrophages in the germ-free mouse

Peritoneal macrophage ultrastructure was analysed stereologically in germ-free mice given a single intraperitoneal injection of sterile, pyrogen-free saline. Thus the stimulant was non-particulate, non-antigenic and inorganic, and effects of immune reactions were minimal. Macrophages were recovered 1, 6, 24 and 72 h after stimulation. A sequence of structural alterations is reported which may be fundamental to macrophage activation. The plasma membrane and nuclear envelope increased in area within only 1 h of saline injection. During the next 5 h loss of plasma membrane, probably by pinocytosis, caused cellular "rounding" and clear-cut alteration in surface configuration. At the same time lysosome-like granules enlarged but decreased in number. By 24 h most cellular structures and compartments (including the plasma membrane) were enlarged. Morphological evidence of nuclear activation accompanied a rather modest enlargement of the nucleus at this stage. The RER hypertrophied last and must, therefore, be judged sufficient in resident macrophages to support the initial growth response which results after stimulation. Thus hypertrophy was observed eventually in every structure examined. Even the minimally activated macrophages resident in the peritoneum of germ-free mice respond readily to stimulation.     

Phosphatidylserine-mediated phagocytosis of influenza A virus-infected cells by mouse peritoneal macrophages

Influenza virus induces apoptosis in cultured cell lines as well as in animal tissues. HeLa cells were infected with influenza virus A/Udon/72 (H3N2) under conditions resulting in almost 100% infection. Such cells underwent typical caspase-dependent apoptosis and were efficiently phagocytosed by macrophages prepared from peritoneal fluids of thioglycolate-treated mice. The membrane phospholipid phosphatidylserine appeared on the surfaces of virus-infected cells at around the time efficient phagocytosis became detectable. In fact, the phagocytosis was almost completely inhibited in the presence of liposomes containing phosphatidylserine, which did not influence the antibody-dependent uptake of zymosan particles by the same macrophages. These results indicate that macrophages phagocytose influenza virus-infected HeLa cells in a manner mediated by phosphatidylserine that appears on the surfaces of infected cells during the process of apoptosis.     

Whole-cell-mount cytochemistry by the colloidal gold labeling method. Combined transmission and scanning electron microscopic study of ConA binding sites in mouse macrophages

Binding, redistribution, and endocytosis of colloidal gold (CG)-labeled concanavalin A (ConA) were studied by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Mouse peritoneal macrophages were cultured on Formvar-coated platinum grids. Either fixed or unfixed cells were labeled by the indirect ConA-CG labeling method. Specimens were critical-point-dried and observed by TEM and SEM in the same region. Surface-bound ConA-CG was easily seen by SEM. Stereomicroscopic observation by TEM clearly showed the three-dimensional distribution of ConA on the cell surface as well as in the cytoplasmic vesicles and vacuoles. In the prefixed cells, CG was distributed randomly on the cell surface. When unfixed cells were labeled at 0 degree C, a similar binding pattern was observed, although the density of bound CG was decreased. When cells labeled with ConA-CG at 0 degree C were further incubated at 37 degrees C, redistribution and endocytosis of the label were seen. Endocytosed CG in the cytoplasmic vesicles and vacuoles was clearly seen by TEM. In addition, three-dimensional location and relationship with other organelles were easily observed. Combined TEM and SEM observation of CG-labeled whole-cell-mount specimens is a useful method to study the dynamics of cell-bound ligands.     

Complement proteins and macrophages. 1. Quantitative estimation of factor B produced by mouse peritoneal macrophages

Cobra venom factor was used for the detection of factor B synthesized by mouse peritoneal macrophages. This method was shown to be specific for factor B assay by neutralization by antimouse factor B antibody. The amount of factor B in the culture supernatant, assessed by this method, was found to be dependent on the medium used for cultivation of macrophages. The addition of 25% L cell-conditioned medium to minimal essential medium (LCM-MEM) enhanced the production of factor B and also of lysozyme. Kinetic analysis in LCM-MEM showed that factor B produced by 6 x 10(4) cells/cm2 increased up to 72 hr and reached a plateau at 96 hr. The amounts of factor B and lysozyme produced in LCM-MEM depended upon the number of macrophages. Production of factor B was completely inhibited by 1 microgram of cycloheximide per ml and was restored by its removal.     

Activation of mouse peritoneal macrophages by synthetic glyceroglycolipid liposomes

Summary Liposomes composed of chemically synthesized glyceroglycolipids, such as 1,2-dipalmityl-[-cellobiosyl-(1 3)]-glycerol (Cel-DAG), 1,2-dipalmityl-[-lactosyl-(1 3)]-glycerol, or 1,2-dipalmityl-[-maltosyl-(1 3)]-glycerol, were found to enhance protective immunity against transplantable tumor cells (sarcoma 180) in ICR mice. Peritoneal exudate cells prepared from mice treated in vivo with Cel-DAG showed cytostatic activity in vitro against the mouse leukemia cell line, EL-4. Adherent cells separated from this preparation showed similar activity. Peritoneal cells from polypeptone-injected mice acquired appreciable cytostatic activity when incubated in vitro in the presence of glyceroglycolipid liposomes. The adherent cell fraction alone showed rather weak cytostatic activity when pretreated with the glyceroglycolipids, and full activity was restored by supplementing with the nonadherent cell fraction. The ability of glycolipids to induce tumoricidal effects was affected by cholesterol content: with increasing cholesterol content, the activities decreased. Cholesterol-free glycolipid liposomes were taken more efficiently by macrophages than cholesterol-containing liposomes. Cholersterol modifies the surface property of glyceroglycolipid liposomes. Activation of macrophages is responsible for enhancement of protective immunity against tumor cells by injection of these glycolipids in vivo.This work was supported in part by Grants-in-Aid (Nos. 58010010, and 59870076) for Scientific Research from the Ministry of Education, Science and Culture of Japan     

Metabolism of exogenous arachidonic acid by mouse peritoneal macrophages

On incubation of resident mouse peritoneal macrophages with arachidonic acid several hydroxyacyl derivatives detectable in cellular supernatants are formed. As main products monohydroxyarachidonic acids (monoHETE's) were identified. In addition, smaller amounts of dihydroxyarachidonic acids (diHETE's) supernatants by reversed phase HPLC, normal phase HPLC in combination with UV-spectroscopy and combined gas-chromatography / masspectrometry revealed the presence of 5-, 8-, 12- and 15 - mono-HETE's, two distinct 5, 12-diHETE's, several 8, 15-diHETE's and 14, 15-diHETE. Among the 5, 12-diHETE's, only small amounts of a compound with the characteristics of LTB, were detected. Under the conditions employed, the cycloxygenase products PGE₂ and PGI₂ (as 6-keto-PGF_1g) were only minor metabolites. In contrast, when macrophage cultures were stimulated with the phagocytic stimulus zymosan, PGI₂, PGE₂ and LTC₄ were found as the major conversion products of arachidonic acid, whereas mono- and diHETE's were not formed in detectable amounts.