Sudden termination of G-CSF injection leads to apoptosis of a large proportion of increased granulocytes.

The effectiveness of granulocyte colony-stimulating factor (G-CSF) for granulocytopenia has been widely recognized. However, although granulocyte counts rapidly increase after a few injection of G-CSF, a large proportion of the increased granulocytes disappear from peripheral blood within a few days following G-CSF withdrawal. Where do they go? In this report, the answer can be seen at a glance. Using MitoCapture and a CCD camera-equipped fluorescence microscope, we succeeded in demonstrating that G-CSF withdrawal induced loss of mitochondrial membrane potential, i.e., an early stage of apoptosis, in human peripheral granulocytes increased by G-CSF.

Suppressive effect of rebamipide, an antiulcer agent, against activation of human neutrophils exposed to formyl-methionyl-leucyl-phenylalanine.

Rebamipide, an antiulcer agent, has been shown to be able to prevent gastric mucosal injury resulting in part from activation of neutrophils. The mechanism of its suppressive action, however, remains to be established. The present study aimed to determine the effect of rebamipide on activation of isolated human neutrophils and to identify the signal transduction pathway involved in its regulation. In unstimulated cells, alkaline phosphatase activity was found residing in short rod-shaped intracellular granules. Upon stimulation with a chemotactic peptide formyl-methionyl-leucyl-phenylalanine, the granules fused to form elongated tubular structures and spherical vacuoles. Rebamipide inhibited reorganization of alkaline phosphatase-containing granules along with upregulation of alkaline phosphatase activity and CD16, a marker of the granules. It also suppressed chemotaxis, an increase in intracellular calcium ion concentration, and NADPH oxidase activation in cells stimulated with formyl-methionyl-leucyl-phenylalanine. In contrast, the drug showed no inhibitory action toward upregulation of alkaline phosphatase activity and CD16, and activation of NADPH oxidase in cells stimulated with phorbol myristate acetate, an activator of protein kinase C. These findings demonstrate that rebamipide exerts a broad spectrum of suppressive actions toward biological functions of human neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine, but not with phorbol myristate acetate, and suggest that the upstream point of protein kinase C is the signal transduction pathway involved in its regulation.

Ultracytochemical study on the localization of superoxide producing sites in stimulated rat neutrophils.

Superoxide anion production in neutrophils plays an important role in the microbicidal defense system in the body. In this study, isolated rat neutrophils were stimulated experimentally and examined by electron microscopy to determine the site of superoxide production and its subsequent translocation during different cell stimulation time periods. Blood and peritoneal neutrophils were incubated for periods of 5, 10, and 15 min with phorbol 12-myristate 13-acetate (PMA), N-formyl-Met-Leu-Phe (fMLP), and combinations of PMA and cytochalasin B (CB) and fMLP and CB. Ultracytochemical detection of O(2)(-) was performed with the 3, 3'-diaminobenzidine-manganese (DAB/Mn) cytochemical method and cationized ferritin (CF) particles were added to stimulation media to monitor endocytotic events that occurred during neutrophil stimulation. Unstimulated neutrophils were devoid of O(2)(-) activity in cytoplasmic granules and at the plasma membrane surface. After 5 min stimulations with PMA, PMA + CB, or fMLP + CB, electron-dense DAB/Mn reaction product was detected in small, centrally located tubular compartments within the neutrophils. CF particles which were added to the stimulation media became internalized in endocytotic vesicles after 5 min stimulation; these vesicles were devoid of O(2)(-) activity. At 10 min stimulation with PMA, O(2)(-)-positive granules subsequently fused with each other and translocated to sub-plasma membrane regions where they either contacted the plasma membrane or fused with CF-containing endocytotic vesicles. Little reaction product was observed on the surface of the neutrophils. Spectrophotometric comparison of the stimulatory effects of PMA, fMLP, and fMLP + CB revealed different rates and yields of O(2)(-) production. Results from this study suggest that the O(2)(-)-producing sites of rat neutrophils originate intracellularly and translocate to the plasma membrane surface following stimulation with PMA, PMA + CB, and fMLP + CB, but not with fMLP or CB alone. Furthermore, these compartments appear to possess the ability to fuse with endocytotic vesicles, a process that may be linked to intracellular microbicidal activity in circulating and tissue neutrophils.

Detection of oxidant producing-sites in glutaraldehyde-fixed human neutrophils and eosinophils stimulated with phorbol myristate acetate.

The aim of the present study was to detect oxidant-producing sites, and to elucidate their dynamic reorganization in human polymorphonuclear leukocytes (PMNs) fixed with glutaraldehyde which preserves cell structure. In biochemical analyses, the detectable O2- generation in unfixed PMNs upon stimulation with phorbol 12-myristate 13-acetate (PMA) in the presence of cytochalasin B was characterized by a lag period of approximately 10 sec followed by O2- production. The maximal rate reached was 3.18+/-0.07 nmol/min/l x 10(6) cells (mean+/-S.D.; n = 4) after 30 sec of stimulation. PMNs exposed to PMA and cytochalasin B followed by fixation with glutaraldehyde generated O2- without a lag period at a rate of 0.35+/-0.05 n mol/min/l x 10(6) cells (mean+/-S.D.) by the addition of NADPH as substrate to the cell suspension. In the cytochemical assays, we employed both cells exposed to PMA and cytochalasin B, and then fixed with glutaraldehyde followed by incubation in the cytochemical reaction medium (pre-fixed cells) and cells incubated in the medium containing PMA and cytochalasin B followed by fixation with glutaraldehyde (post-fixed cells). Oxidant reaction in the pre-fixed cells was detected by the addition of NADPH and FAD to the reaction medium. No oxidant-reaction product was seen in pre-fixed cells stimulated for 10 sec whereas the oxidant reaction was visualized in intracellular compartments of pre-fixed PMNs stimulated for 20 sec. The fact that the pre-fixed PMNs stimulated for 30 sec showed increased numbers of oxidant-producing structures compared to those seen in the pre-fixed cells stimulated for 20 sec, demonstrates that the amount of the reaction product and the number of oxidant-producing intracellular compartments increases between 20 and 30 sec after start of stimulation with PMA. These cytochemical results using the pre-fixed cells coincided with the findings obtained from the biochemical assays in the pre-fixed cells exposed to PMA and cytochalasin B. The oxidant reaction was observed in elongated tubular structures that were arranged in a radial fashion, and were associated with the plasma membrane in the pre-fixed PMNs, whereas post-fixed PMNs exhibited slender spherical or rod-shaped structures of various lengths. The present results indicate that the pre-fixed PMNs can be employed for elucidating the dynamic reorganization of oxidant-producing sites in human PMNs.

An ultracytochemical study on the dynamics of alkaline phosphatase-positive granules in rat neutrophils.

Alkaline phosphatase (ALPase) activity was examined by cerium-based ultracytochemistry in isolated rat neutrophils following stimulation with phorbol myristate acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP). In control neutrophils, low levels of ALPase activity were detected in small tubular and spherical compartments distributed throughout the cytoplasm. Neutrophils stimulated for 2.5, 5, 15, and 30 min with 50 ng/ml PMA or 10(-7) M fMLP displayed a time-dependent increase in ALPase activity. At 2.5 min, an increase in activity was first identified in compartments that were aggregated in the central regions of the cell. By 15 min, a dense precipitate was seen in tubular or elongated bead-like structures that extended to and made contact with the plasma membrane. Large enzyme-positive vacuoles were also observed in regions near the plasma membrane. At the longer stimulation times, a fine precipitate was present on the cell surface of the neutrophil in regions where subplasmalemmal ALPase activity was present. The results of this study indicate that an increase in activity and a redistribution of ALPase-positive structures occurs in neutrophils in response to stimulation with PMA and fMLP. It is likely that these compartments are latent pools of ALPase which, upon stimulation, fuse and mobilize the enzyme activity to the cell surface.

Internalization of ecto-ATPase activity in human neutrophils upon stimulation with phorbol ester or formyl peptide.

We have demonstrated the alteration of the localization of ecto-ATPase activity in human neutrophils after stimulation with phorbol myristate acetate or N-formylmethionyl-leucyl-phenylalanine using a cerium-based cytochemical method. In unstimulated cells, the enzyme activity was observed on the plasma membrane. Both the diazonium salt of sulfanilic acid and diethylpyrocarbonate inhibited the production of the reaction precipitates. Within 2-3 min of stimulation, cells developed cytoplasmic projections (ruffles). The ecto-ATPase activity on the plasma membrane of these ruffles was, however, weaker than that at the non-ruffle-forming side. The ruffle-forming side (RFS) was also the site where elongated tubular structures positive for the enzyme reaction tended to concentrate and associated with the plasma membrane. The enzyme activity was also detected in intracellular compartments, which appeared predominantly in the RFS concomitantly with the disappearance of the enzyme activity from the plasma membrane. Using a series of thick sections and computer-assisted three-dimensional reconstruction, the enzyme reaction-positive internalized membranes were visualized as a complicated mass formed by enzyme reaction-positive vesicles which gathered together and were, at least in part, interconnected. The present results indicate that the detected enzyme reaction is a product of the ecto-ATPase activity, and that RFS possibly serves the membrane flow with respect to endocytosis.

Biochemical properties and cytochemical localization of ouabain-insensitive, potassium-dependent p-nitrophenylphosphatase activity in rat atrial myocytes.

Enzyme activity that represents ouabain-insensitive, potassium-dependent p-nitrophenylphosphatase (p-NPPase) was assessed in rat atrial myocytes by biochemical and cytochemical procedures. No activity was detected in parallel experiments with ventricular myocytes. Fixed tissues were incubated in a reaction medium containing Tricine buffer, p-nitrophenylphosphate (p-NPP), KCl, MgCl2, CaCl2, CeCl3. Triton X-100, levamisole, and ouabain. Final pH was adjusted to 7.5. Biochemical studies showed that accumulation of p-nitrophenol in the medium was increased proportionally in accordance with the amount of incubated tissue. This activity was optimal with incubation at pH 7.5 and in the presence of KCl. Approximately 70% of the enzyme was inhibited by 2 mM CeCl3. Electron microscopic observations revealed reaction product (RP) at sites of ouabain-insensitive, potassium-dependent p-NPPase activity as electron-dense precipitate localized at the inner surface of the plasma membrane and at the T-tubules of atrial myocytes. Control experiments indicated that the activity was strongly inhibited by sodium orthovanadate and was repressed by omeprazole and 1,3-dicyclohexylcarbodiimide. X-ray microanalysis confirmed the presence of cerium within the cytochemical RP. The ouabain-insensitive, K-dependent p-NPPase activity detected in the present study is considered to be an isoform of a P-type, H-transporting, K-dependent adenosine triphosphatase (H,K-ATPase).

Ultracytochemical localization of alkaline phosphatase (ALPase) activity in neutrophils of the rat lung following injection of lipopolysaccharide.

Alkaline phosphatase (ALPase) activity in neutrophils was examined by enzyme ultracytochemistry at sites of experimentally induced acute inflammation in the rat lung and compared with those in non-inflammatory regions. Neutrophil accumulations in the lung were stimulated by intraperitoneal (IP) injection and intratracheal (IT) instillation of lipopolysaccharide (LPS), an endotoxin from Escherichia coli. Microsliced sections of fixed lung were incubated in a cerium-based reaction medium for the detection of ALPase activity. As cytochemical controls, sections were incubated in a substrate-free medium or in a medium containing 2 mM levamisole for inhibition of ALPase activity. Both IP and IT injections resulted in a significant accumulation of neutrophils in the lung, however, their histological distributions differed, the former stimulating high accumulations within the capillaries and interalveolar spaces, and the latter within the confines of the alveolar spaces. In neutrophils from controls and non-inflamed regions of the lung, ALPase activity was detected as an electron-dense reaction product localized predominantly to small spherical and tubular membrane-bounded cytoplasmic compartments. In the IP-injected rats, prominent ALPase activity was observed at their plasma membrane surfaces, most notably at sites of endothelial cell contact. Following IT instillation of LPS, neutrophils suspended in the alveolar spaces showed a reduced plasma membrane reactivity, however, at type I pneumocyte contact regions, enzyme activity was significantly increased. In all cases, ALPase activity was not detected at the endothelial plasma membranes. Some reaction, however, was seen on the microvilli of the type II surfactant-producing cells. These results indicate that ALPase activity of rat neutrophils in the lung can be increased by LPS injection and that its activity may also be related to the sites of cell-cell interaction and cell surroundings.

Ultracytochemical study of trimetaphosphatase activity during acrosomal formation in the mouse testis.

The localization of Trimetaphosphatase (TMPase) activity during the acrosomal formation in the mouse testis was enzyme cytochemically investigated by the cerium-salt method. In addition to the lysosomes of the Sertoli cells and the spermatogenic cells in the seminiferous tubules, positive TMPase activity was detected in the Golgi complex and in the acrosomal vesicles of the spermatids, as well as in the acrosomes of both spermatids and spermatozoa. In the Golgi complex of the spermatids, TMPase activity was observed in the first one or two lamellae of the trans-face and in the small vesicles in the vicinity of the Golgi complex. TMPase positive reaction was also detected in the acrosomes of the spermatozoa in the lumina of both the seminiferous tubules and the epididymal duct. The localization of this enzyme activity was compared with that of acid phosphatase (ACPase), as detected by the cerium-based method, using beta-glycerophosphate as substrate: ACPase activity was completely absent from the Golgi complex, small vesicles, acrosomal vesicle and acrosome throughout the entire process of acrosomal formation. TMPase is thought to become one of the acrosomal components, and may be involved in the acrosomal reaction during fertilization.

Na-K-ATPase activity in the guinea pig stria vascularis in experimentally-induced endolymphatic hydrops.

The effect of endolymphatic hydrops on the Na-K-ATPase activity in the guinea pig stria vascularis was electron microscopically and enzyme cytochemically investigated one year after experimental induction. The morphological observations revealed intercellular dropsy in the basal infoldings of the marginal cells, and shrinkage and disappearance of intermediate cells. Moreover, shrinkage of the marginal cells, especially of the basal infoldings, was occasionally observed. In spite of these morphological alterations, the Na-K-ATPase activity was still detected on the plasma membrane of the basal infoldings of most marginal cells. No remarkable differences were found among the cochlear turns of the specimens examined. However, no reaction product was detected on the basolateral plasma membrane of severely degenerated marginal cells. The present results indicate that the Na-K-ATPase of the plasma membrane of the basal infoldings of the marginal cells plays an important role in the maintenance of the unique ion concentration of the endolymph even in the endolymphatic hydropic condition, and that the Na-K-ATPase activity is attenuated in severely atrophic cells.

Alkaline phosphatase, 5'-nucleotidase and magnesium-dependent adenosine triphosphatase activities in the transitional epithelium of the rat urinary bladder.

The cerium-based method was used to demonstrate cytochemically the ultrastructural localization of alkaline phosphatase (ALPase), 5'-nucleotidase (5'-Nase) and magnesium-dependent adenosine triphosphatase (Mg-ATPase) on the transitional epithelium of the rat urinary bladder. The reaction product for ALPase was found on the plasma membrane of all epithelial cells, except the luminal surface of superficial cells. The activity of 5'-Nase appeared on the plasma membrane of all bladder transitional epithelial cells, including the free surface of superficial cells. The Mg-ATPase reaction product was seen on the plasma membrane of superficial, intermediate and basal cells, but never on the luminal surface of superficial cells and it was only occasionally seen on the basal surface. The possible functions of these phosphatases have been discussed, and it was emphasized that the 5'-Nase activity present on the luminal surface of superficial cells may play a special role in the membrane movement of these cells in the transitional epithelium.

Ultracytochemical localization of acid phosphatase and trimetaphosphatase activities in the transitional epithelium of the rat urinary bladder.

Acid phosphatase and trimetaphosphatase activities have been demonstrated cytochemically in the transitional epithelium of the rat urinary bladder. Acid phosphatase activity was examined by the method of Robinson and Karnovsky (1983), and trimetaphosphatase activity was examined by the method of Kobayashi et al. (1988). Intense positive reaction for acid phosphatase activity was observed with both beta-glycerophosphate and p-nitrophenyl phosphate as substrates, in lysosomes, Golgi complex, GERL, multi-vesicular bodies and wrapping lysosomes. The reaction product of the trimetaphosphatase activity was visualized as fine particulated deposits along the limiting membrane of some lysosomes, and of tubular structures located in the basal cytoplasm. There seems to be a fine distinction and reciprocal complementary functional relationship between two kinds of lysosomes containing either acid phosphatase or trimetaphosphatase activity.

Carbohydrates in the guinea pig stria vascularis demonstrated with lectin-gold techniques.

Soybean agglutinin (SBA), Helix pomatia agglutinin (HPA), Ricinus communis agglutinin-II (RCA-II), Limax flavus agglutinin (LFA) and wheat germ agglutinin (WGA) were employed to determine the localization of specific carbohydrates on thin sections of lowicryl K4M embedded guinea pig striae vasculares using the lectin-gold and glycoprotein-gold techniques. SBA, HPA and RCA-II gold labeling was observed in many of the cytoplasmic vesicles, endosomes and apical tubules located in the supranuclear region as well as on the microvilli and micropinocytotic invaginations of the luminal plasma membrane of the marginal cells. LFA labeling was found on the basal plasma membrane of the marginal cells as well as in the basement membrane of the perivascular spaces. WGA binding sites were detected along the plasma membrane of all types of cells constituting the stria vascularis. Our present results revealed that the membranes of internalization and many of the cytoplasmic vesicles, endosomes and apical tubules in the supranuclear region of the marginal cells are associated together and it is suggested that these structures may be related to the regulation of endolymph.