ELECTRON MICROSCOPE OBSERVATIONS ON COMPOUND 48/80-INDUCED DEGRANULATION IN RAT MAST CELLS : Evidence for Sequential Exocytosis of Storage Granules

In vitro degranulation of rat mast cells was studied at different intervals ranging from 10 to 60 sec after adding the histamine liberator, compound 48/80 (0.4 µg/ml, 17°C). The ultrastructural changes were followed by electron microscopy, and parallel assays were made to determine the histamine released. In addition, the extracellular tracers lanthanum and hemoglobin (demonstrated by its peroxidative activity) were applied to mast cells to follow communication of the extracellular space with the cavities formed during degranulation. After a lag period of 10 sec, degranulation started in the most peripherally located granules. The perigranular membrane fused with the plasma membrane, resulting in a pore bridged by a thin diaphragm. This was followed by rupture of the diaphragm and extrusion of the granule matrix (exocytosis). The process advanced towards the cell interior by fusion and opening of the deeper situated granules to the formerly opened granule cavities. At the end of the process, the cell was filled by a system of complicated cavities containing a number of altered granules. Extracellular tracers have shown that these intracellular cavities were in unbroken communication with the extracellular space from the very beginning of their formation. Both lanthanum and hemoglobin were found to be adsorbed to the limiting membrane of the cavities and bound to altered mast cell granules. In contrast, no tracer substance was present in nondegranulating mast cells. Degranulation of mast cells by compound 48/80 is regarded as a sequential exocytosis, a process similar to that described for some exocrine gland cells. All the "intracellular" cavities, formed by degranulation, were shown to communicate with the extracellular space; consequently, granules lying in these cavities must be considered as biologically extracellular. The present findings support the view that histamine is released from the granule matrix by the extracellular ionic milieu.     

Surface morphology of rat peritoneal mast cells during in vitro regeneration after histamine secretion

Summary Cell-surface morphology of regenerating mast cells was followed over a period of 48 h after histamine release. Control cells (not stimulated to secrete) were characterized by anastomosing folds of membrane of equal depth and width. During exocytosis these folds disappeared and were replaced by deep cup-shaped flaps of membrane evident in cells incubated for 10 min. During the first hours of regeneration these flaps fused mutually or with the plasma membrane. This activity suggests membrane retrieval, maybe specifically recycling the granule-type patches of membrane. Membrane-fusion activity was observed to some degree also after extended incubation. After 48 h of incubation the regeneration process was still not completed, as indicated by the fact that holes leading to intracellular cavities could still be found.     

Early membrane retrieval following exocytosis in rat mast cells

The fate of the surplus membrane following exocytosis of mast cell granules was studied by the extracellular tracer Ruthenium red (Ru red). Isolated rat peritoneal mast cells were stimulated with 4 micrograms/ml polylysine, washed and maintained in a culture medium for 80 min. Mast cells were observed both with the light microscope after adding Ru red in physiological solution and with the electron microscope after fixation in Ru red-containing fixatives. Whereas all exocytotic cavities were found to be stained with Ru red immediately after stimulation, a gradual lack of staining was observed in the subsequent period. The exits of the cavities were sealed by membrane fusions which resulted in closed vacuoles containing exocytosed granule remnants. These vacuoles often fused with each other to form a few giant vacuoles. The overwhelming majority of the vacuoles were observed to be closed 30 to 80 min after stimulation. In one experiment a quantitative analysis was performed to assess the degree of membrane recapture by sealing of the exocytotic cavities. A considerable portion of the plasma membrane area was retrieved in this way as early as between 15 and 30 min after stimulation. We conclude that the dominant mechanism of membrane retrieval in the early period following exocytosis is the recapture of large membrane areas by sealing of exocytotic cavities.     

Toxoplasma gondii and mast cell interactions in vivo and in vitro: experimental infection approaches in Calomys callosus (Rodentia, Cricetidae)

In the present work, we studied some qualitative and quantitative characteristics of mast cells located in the peritoneal cavity, submandibular and dorsal lymph nodes and ileum of Calomys callosus experimentally infected by Toxoplasma gondii. In uninfected animals, the majority of mast cells had similar ultra-structural characteristics, including several cytoplasmic granules with homogeneous and electron dense contents. However, after 1 h of infection, a significant influx of mast cells into peritoneal cavity was observed. The number of mast cells in this compartment decreased progressively in infected animals, and was significantly lower than the number of mast cells in control animals after 48 h of infection. Mast cells from infected animals or from purified suspensions that were infected in vitro presented significant morphological modifications, suggesting a degranulation process: cytoplasmic granules with electron dense content, fusion of the cytoplasmic granules, intracytoplasmic channels, cytoplasmic granules with flocculent material, plasma membrane rupture and granule contents in the extracellular environment. A remarkable increase in the influx of neutrophils toward the peritoneal cavity of the infected animals was observed after 12 h of infection. Moreover, this event occurred after the mast cell degranulation process took place. The relative increase in the number of mast cells and neutrophils was also followed by an increase in the number of macrophages, but there was a significant decrease in lymphocyte influx. After 48 h of infection, the parasite had spread from the peritoneal cavity to all organs examined. Also, mast cells from these organs showed evident morphological alterations, indicating the presence of the degranulation process. These results suggest that mast cells are deeply involved with the acute phase of the inflammatory response in this experimental model.     

Proliferative potential of murine peritoneal mast cells after degranulation induced by compound 48/80, substance P, tetradecanoylphorbol acetate, or calcium ionophore A23187.

Proliferative potential of degranulated mast cells was investigated. Mast cells were collected from the peritoneal cavity of mice, and degranulation was induced by compound 48/80, substance P, 12-O-tetradecanoylphorbol 13-acetate (TPA), or calcium ionophore A23187. The potentiality of colony formation in methylcellulose was not reduced by treatment of various concentrations of compound 48/80, substance P and TPA. When degranulation was induced by compound 48/80, substance P or TPA, proportion of highly degranulated mast cells containing less than five granules was rather small. In contrast, considerable proportion of highly degranulated mast cells was obtained after the treatment with the low concentration (0.1 microgram/ml) of A23187. These highly degranulated mast cells, which were individually picked up by the micromanipulator, proliferated not only in methylcellulose but also in the skin of mast cell-deficient WBB6F1-W/Wv mice. Inasmuch as we have already shown the proliferation of IgE-sensitized and Ag-stimulated mast cells, degranulated mast cells appear to retain the proliferative potential in general.     

Identification of P-glycoprotein at the membrane of mast cell secretory granules. An immunofluorescence and protein A-gold electron microscopical investigation

The presence of P-glycoprotein has been investigated in rat peritoneal mast cells by means of immunofluorescence and immunogold electron microscopy, using the specific monoclonal antibody JSB-1. Immunofluorescence studi es showed that the glycoprotein is primarily concentrated in mast cell granules, and little is localized at the plasma membrane. Electron microscope observations revealed a marked accumulation of colloidal gold particles at the granule-coating membranes, whereas decoration of the plasma membrane is much less intense. When mast cells are stimulated to exocytate with compound 48/80, both immunofluorescence and electron microscopy showed concentration of P-glycoprotein reactivity at the plasma membrane level. Indeed, fusion of the granule with the plasma membrane allowed transfer of immunoreactive P-glycoprotein material from the granule-coating membrane to the cell surface membrane. These findings confirmed the presence of P-glycoprotein in mast cells; it is predominantly localized in the granules and is exposed on the cell surface only after exocytosis, suggesting, therefore, a possible physiological role for P-glycoprotein in the secretion of certain mediators. This revised version was published online in November 2006 with corrections to the Cover Date.     

Identification of P-glycoprotein at the membrane of mast cell secretory granules. An immunofluorescence and protein A-gold electron microscopical investigation

The presence of P-glycoprotein has been investigated in rat peritoneal mast cells by means of immunofluorescence and immunogold electron microscopy, using the specific monoclonal antibody JSB-1. Immunofluorescence studi es showed that the glycoprotein is primarily concentrated in mast cell granules, and little is localized at the plasma membrane. Electron microscope observations revealed a marked accumulation of colloidal gold particles at the granule-coating membranes, whereas decoration of the plasma membrane is much less intense. When mast cells are stimulated to exocytate with compound 48/80, both immunofluorescence and electron microscopy showed concentration of P-glycoprotein reactivity at the plasma membrane level. Indeed, fusion of the granule with the plasma membrane allowed transfer of immunoreactive P-glycoprotein material from the granule-coating membrane to the cell surface membrane. These findings confirmed the presence of P-glycoprotein in mast cells; it is predominantly localized in the granules and is exposed on the cell surface only after exocytosis, suggesting, therefore, a possible physiological role for P-glycoprotein in the secretion of certain mediators. This revised version was published online in November 2006 with corrections to the Cover Date.     

The protective effect of prolil-glycil-proline peptide (PGP) under compound 48/80-induced anaphylactoid reaction in mice

The influence of PGP on compound 48/80-induced anaphylactoid reaction development in mice and on histamine secretion from rat peritoneal mast cells (RPMS) under their activation by compound 48/80 were investigated. Anaphylactoid reaction was caused by intraperitoneal injection of compound 48/80 into mice. The number of animals with manifestations of anaphylactoid reaction symptoms, the severity of these symptoms, the amount of died animals and the time of death were registering during an hour. Mast cells for in vitro investigations were obtained from rats’ peritoneal cavity. Secreted histamine was evaluated from formation of fluorescent product of it’s condensation with ortho-phthalaldehyde. The preventive injection of PGP in mice (15 min before compound 48/80) decreased the mortality rate of animals and intensity of anaphylactoid reaction symptoms. But PGP had no effect on histamine secretion from mast cells under their activation by compound 48/80 in vitro. Results show that there is a component in the mechanism of PGP protective effect under anaphylactoid reaction which is not connected with mast cells stabilization.     

Visualization of exocytotic secretory processes of mast cells by fluorescence techniques

Secretory processes via exocytosis in rat peritoneal mast cells were visualized by two complementary fluorescence techniques; one staining pre-exocytotic granules with a basic probe and the other staining post-exocytotic granules with acidic probes. Granules within mast cells were selectively stained with acridine orange and emitted orange yellow fluorescence. Upon stimulation with compound 48/80, release of acridine orange from granules was observed both in population and single cell measurements. This release was seen in some localized area of mast cells. Opening of pores between plasma membranes and granule membranes was monitored using acidic fluorescence probes such as 6-carboxyfluorescein or lucifer yellow CH. Not only granules located at peripheral region, but also granules near the core region participated in exocytosis. The existence of junctions between these granules was suggested. TMA-DPH, a lipophilic membrane probe, which was localized at plasma membrane before stimulation, diffused into granule membranes after stimulation. This shows that after stimulation, some constituents of plasma and granule membranes were mixed. Even after extensive degranulation, mast cells extruded acidic probes, indicating the plasma membranes still play a role of barrier. Activation of lateral motion of granules preceding to exocytosis was not observed. It was concluded that the visualization of secretory processes by fluorescence and image processing techniques will be useful for the study of molecular mechanisms underlying exocytosis.     

Morphological alteration of peritoneal mast cells and macrophages in the mouse peritoneal cavity during the early phases of an allergic inflammatory reaction

We investigated the presence of mast cell granules in macrophages following an in vivo model of an allergic reaction. Injection of ovalbumin (100 microg) into the peritoneal cavity of sensitised mice produced a rapid (within 2 h) influx of neutrophils followed by a slower (after >4 h) eosinophil migration. Ovalbumin treatment induced a high incidence (approximately 50%) of mast cell degranulation compared to control phosphated-buffered saline-treated mice. The majority (approximately 90%) of peritoneal macrophages contained mast cell granules as early as 2 h post-ovalbumin, with lower values at later time-points, as determined by staining with Toluidine blue and Berberine sulphate. This was confirmed by electron microscopy which enabled us to identify the complex mast cell granule sub-structural components in macrophage phagosomes. In conclusion, we used histochemical and ultrastructural analyses to show that mast cell granules become internalised with macrophages during the early stages of an experimental allergic reaction.     

Degranulation of eosinophilic granule cells in neurofibromas and gastrointestinal tract in the bicolor damselfish

Damselfish neurofibromatosis (DNF) is a neoplastic disease affecting bicolor damselfish (Stegastes partitus Poey) on Florida reefs. Previous studies have demonstrated high densities of eosinophilic granule containing cells (EGC), the proposed equivalent of mast cells in fishes, in neurofibromas and malignant peripheral nerve sheath tumors (mpnst) in DNF. These lesions are similar to those in the disease neurofibromatosis type 1 (NF1) in humans, which contain large numbers of mast cells. In the present study, experiments were conducted to measure the response of EGC in these tumors as well as in the submucosa of the digestive tract to the mast cell degranulating agent compound 48/80. Degranulation of these cells was visible by light microscopy and characterized by conspicuous swelling of granules and often by the presence of free granules adjacent to the EGC. Degranulation occurred by release of intact granules (diacytosis), as reported in other fishes, rather than by fusion of granules with the cell membrane (exocytosis) as reported in mast cells in mammals. Baseline levels of EGC exhibiting degranulation ranged from 20-26% in the submucosa to 30% in tumors. Within 1-2h of exposure to compound 48/80, significant increases in average levels of degranulation were observed, to 67% in the gut and 72% in tumors. Degranulation was significantly more extensive in the tumors than in the gut. The outermost edges of the tumors contained significantly higher densities of EGC but these cells exhibited lower rates of degranulation than those in the inner regions of tumors. These observations support the hypothesis that the EGC present in neurofibromas and mpnst in DNF are equivalent to the mast cell component in neurofibromas in NF1.     

Morphological study of rat mast cells stimulated with compound 48/80 at different temperatures

Changes of mast cells stimulated with compound 48/80 were morphologically investigated at different temperatures. Peritoneal mast cells of male rats were stimulated in vitro at 4 or 17° C. At 17° C, mast cells stimulated for 10 s gave decreased fluorescent reactions for phalloidin. At 30 s stimulation, they showed typical exocytosis initiated by fusions of peripherally located secretory granules to the plasma membrane. In contrast, mast cells stimulated at 4° C exhibited neither decrease of phalloidin reactions nor typical excytosis even after 30 s. It was inferred that the fusions were mediated by cytoplasmic elements, probably the actin filaments previously suggested to prevent release of secretory granules. Furthermore, the space between the perigranular membrane and granular contents was enlarged in some mast cells stimulated at 4° C. The morphological changes suggested that equivocal events occurred also in the cytoplasm of these cells. The mast cells showed no typical exocytosis at 4° C.     

Membrane retrieval in non-exocytic and exocytic rat peritoneal mast cells

Mast cells were incubated with the membrane marker cationized ferritin (CF) in order to study the fate of cell membrane in both non-exocytic and in exocytic cells. Non-exocytic mast cells took up CF by a macropinocytic process by fusion of microfolds. CF particles occurred in lysosomal-like structures, in mast cell granules, and eventually in Golgi vesicles. Exocytic cells took up CF mainly by micropinocytosis and after 2 h CF appeared in Golgi vesicles and in progranules. The different uptake routes of CF in non-exocytic and in exocytic mast cells probably reflect that the former cells constantly take up material by macropinocytosis, perhaps for nutrition. The latter cells undergo a process of exocytosis-endocytosis in order to re-use retrieved surface membrane in new granule formation and in order to regain their ordinary size, which is greatly increased upon secretion.     

Redistribution of phospholipid/Ca2+-dependent protein kinase in mast cells activated by various agonists

Three types of agonists; receptor-mediated concanavalin A), direct (phorbol ester), and membrane-perturbing (compound 48/80), elicit histamine secretion from rat peritoneal mast cells. We tested whether activation of the mast cells by these agents is accompanied by subcellular redistribution of protein kinase C. Phorbol ester treatment predictably caused a profound decrease of phospholipid/Ca2+-dependent histone kinase activity in the cytosol and a concomitant increase of [3H]PMA-binding capacity in the membrane fraction, in a time- and concentration-dependent manner. Similar, but less marked effects were observed with stimulations by concanavalin A and compound 48/80. When mast cells labeled with [32P] and then stimulated with the agents, phosphorylation of a 50,000-Dalton protein was enhanced in the membrane fraction. These results suggest that protein kinase C may play a role in mast cell activation through phosphorylation of the membrane protein.     

Ultrastructural characteristics of rat peritoneal mast cells undergoing differential release of serotonin without histamine and without degranulation

Summary Rat mast cells pretreated with the tricyclic antidepressant drug amitriptyline and stimulated with compound 48/80 secreted 60% of the total serotonin present in the cells, but only 15% of histamine, another amine stored in the same granules. Ultrastructural studies demonstrated that mast cells undergoing such differential release do not exhibit classical degranulation by compound sequential exocytosis. However, there were changes in granule shape and size, as well as alterations in many morphometric parameters consistent with secretion. Storage granules lost their homogeneity, exhibited greatly reorganized matrix and were surrounded by clear spaces which were often associated with small (0.1–0.01 m) cytoplasmic vesicles, some of which contained electron-dense material. Secretory granules often had bud-like protrusions or were fused together in series. Quantitative autoradiography localized ³H-serotonin outside the storage granules, close to small vesicles, while staining with ruthenium red demonstrated that vesicular structures associated with differential release were not endocytotic. These results suggest that amitriptyline may inhibit regular exocytosis and permit at least serotonin to be moved selectively from storage granules to the cytosol or small vesicles from which it is eventually released.     

Mechanisms of Granule Membrane Recapture following Exocytosis in Intact Mast Cells

In secretory cells, several exocytosis-coupled forms of endocytosis have been proposed including clathrin-mediated endocytosis, kiss-and-run endocytosis, cavicapture, and bulk endocytosis. These forms of endocytosis can be induced under different conditions, but their detailed molecular mechanisms and functions are largely unknown. We studied exocytosis and endocytosis in mast cells with both perforated-patch and whole-cell configurations of the patch clamp technique using cell capacitance measurements in combination with amperometric serotonin detection. We found that intact mast cells exhibit an early endocytosis that follows exocytosis induced by compound 48/80. Direct observation of individual exocytic and endocytic events showed a higher percentage of capacitance flickers (27.3%) and off-steps (11.4%) in intact mast cells than in dialyzed cells (5.4% and 2.9%, respectively). Moreover, we observed a type of endocytosis of large pieces of membrane that were likely formed by cumulative fusion of several secretory granules with the cell membrane. We also identified “large-capacitance flickers” that occur after large endocytosis events. Pore conductance analysis indicated that these transient events may represent “compound cavicapture,” most likely due to the flickering of a dilated fusion pore. Using fluorescence imaging of individual exocytic and endocytic events we observed that granules can fuse to granules already fused with the plasma membrane, and then the membranes and dense cores of fused granules are internalized. Altogether, our results suggest that stimulated exocytosis in intact mast cells is followed by several forms of compensatory endocytosis, including kiss-and-run endocytosis and a mechanism for efficient retrieval of the compound membrane of several secretory granules through a single membrane fission event.     

Accumulation of low density lipoproteins in stimulated rat serosal mast cells during recovery from degranulation

Stimulation of rat serosal mast cells in vitro with compound 48/80, a degranulating agent, resulted in an immediate increase in binding of low density lipoproteins (LDL) to the stimulated mast cells. The increase in binding was dose-dependent and closely followed the increase in histamine release, i.e., the exocytosis of mast cell granules. It could be demonstrated that the LDL were bound to exocytosed secretory granules which remained cell-associated. During the recovery period the granule-bound LDL were internalized by the mast cells along with the granules. A single stimulation of mast cells rendered their cytoplasm to be filled with granular material showing positive staining for both apoB and neutral lipid. This change was accompanied by a 30-fold increase in the cellular content of cholesteryl esters. Thus, rat serosal mast cells possess a specific mechanism for uptake of LDL that is activated by stimuli that lead to degranulation, the result being massive uptake of LDL by stimulated mast cells during recovery from degranulation.     

Mast cell degranulation and its inhibition by an anti-allergic agent tranilast. An electron microscopic study

Degranulation of IgE-sensitized rat mast cells by antigen was studied quantitatively in vitro and in vivo by electron microscopy. The inhibition of this degranulation by an anti-allergic drug, N-(3,4-dimethoxycinnamoyl)anthranilic acid (Tranilast), was also examined both in vitro and in vivo. In the in vitro study using peritoneal mast cells, alteration of the granules, cavity formation by fusion of the perigranular membrane and granule discharge due to fusion of the cavity membrane with the cell membrane were observed and were accompanied by histamine release. Scanning electron microscopy disclosed the extrusion of smooth, round bodies from pores formed on the cell surface. In the in vivo study of passive cutaneous anaphylaxis (PCA), the characteristic features of mast cell degranulation were obvious 5 min after the injection of antigen; leakage of dye increased progressively from 5 to 30 min but was not found at 6 h. From quantitative analysis of the substructure of mast cells, it was demonstrated that degranulation of IgE-sensitized mast cell induced by antigen was achieved by sequential exocytosis both in vitro and in vivo. Tranilast inhibited these changes to a remarkable extent and it was concluded that the inhibition of mast cell degranulation by this drug might play an important role in anti-allergic treatment.     

Phospholipases D1 and D2 regulate different phases of exocytosis in mast cells

The rat mast cell line RBL-2H3 contains both phospholipase D (PLD)1 and PLD2. Previous studies with this cell line indicated that expressed PLD1 and PLD2 are both strongly activated by stimulants of secretion. We now show by use of PLDs tagged with enhanced green fluorescent protein that PLD1, which is largely associated with secretory granules, redistributes to the plasma membrane in stimulated cells by processes reminiscent of exocytosis and fusion of granules with the plasma membrane. These processes and secretion of granules are suppressed by expression of a catalytically inactive mutant of PLD1 or by the presence of 50 mM 1-butanol but not tert-butanol, an indication that these events are dependent on the catalytic activity of PLD1. Of note, cholera toxin induces translocation of PLD1-labeled granules to the plasma membrane but not fusion of granules with plasma membrane or secretion. Subsequent stimulation of calcium influx with Ag or thapsigargin leads to rapid redistribution of PLD1 to the plasma membrane and accelerated secretion. Also of note, PLD1 is recycled from plasma membrane back to granules within 4 h of stimulation. PLD2, in contrast, is largely confined to the plasma membrane, but it too participates in the secretory process, because expression of catalytically inactive PLD2 also blocks secretion. These data indicate a two-step process: translocation of granules to the cell periphery, regulated by granule-associated PLD1, and a calcium-dependent fusion of granules with the plasma membrane, regulated by plasma membrane-associated PLD2 and possibly PLD1.     

Low density lipoprotein degradation by rat mast cells. Demonstration of extracellular proteolysis caused by mast cell granules

The interaction between rat serosal mast cells and low density lipoproteins (LDL) was studied in vitro. When rat 125I-LDL was incubated with mast cells, it was bound to a binding site on the mast cell surface but was not internalized by the cells. Even though 125I-LDL was not internalized, its protein component, apolipoprotein B, was rapidly degraded. The proteolytic activity responsible for the degradation of apolipoprotein B was present in the extracellular fluid of mast cells. It could be shown that the degradation was caused entirely by specific cell organelles of mast cells, the granules, which were spontaneously released into the extracellular fluid during preparation and incubation of the cells. In contrast to uncontrolled spontaneous degranulation, a controlled specific degranulation of mast cells can be induced by treating the cells with the compound 48/80. When increasing amounts of 48/80 were added to mast cell suspensions, a dose-dependent release of granules was observed and an increase in the rate of 125I-LDL degradation resulted. The increase in 125I-LDL degradation closely followed the increase in granule release. Thus, a quantitative relationship between the amount of granules present in the extracellular fluid and the amount of degradation of 125I-LDL could be established. The apolipoprotein part of LDL was extensively degraded by isolated mast cell granules. Analysis by polyacrylamide gel electrophoresis showed that upon incubation of LDL with isolated granules, the apolipoprotein B band rapidly disappeared with simultaneous appearance of several low molecular weight bands. The degradation of 125I-LDL by mast cell granules proceeded optimally at neutral pH and at physiological ionic strength. The results show that mast cell granules are able to efficiently degrade LDL in vitro, once released from mast cells into the extracellular fluid.