Single Mast Cells Research Articles

Capillary electrophoretic study of individual exocytotic events in single mast cells

In this work we have demonstrated the application of on-column dynamic release of serotonin from individual granules within rat peritoneal mast cells (RPMCs). These granules are approximately 0.25 fl in volume and represent some of the smallest entities studied by capillary electrophoresis (CE). With the coupling of high-speed CE and laser-induced native fluorescence, a time resolution of 0.002 min and a detection limit of 0.9 amol ( S/N=3, rms) were achieved. By using a secretagogue as the running buffer for CE, we resolved the peak profile of individual degranulation events released due to exocytosis. The average amount of serotonin released for five RPMCs analyzed was 33 amol ±16%.

Monitoring exocytosis and release from individual mast cells by capillary electrophoresis with laser-induced native fluorescence detection.

The complex temporal evolution of on-column exocytotic release of serotonin and proteins from individual rat peritoneal mast cells (RPMCs) was monitored by using capillary electrophoresis. Laser-induced native fluorescence detection with 275-nm excitation was used, and a detection limit of 1.7 amol (S/N = 3; rms) was obtained for serotonin. A physiological running buffer was used to ensure that the cell remained viable throughout. The secretagogue was polymyxin B sulfate (Pmx). Following the injection of a single mast cell into the capillary, electromigration of Pmx toward and past the cell induced degranulation and release of serotonin. The time course of release was registered in the electropherograms with subsecond resolution. Subsequent introduction of SDS caused the cell to lyse completely and allowed the residual serotonin to be quantified. The average amount of serotonin observed per RPMC was 1.6 +/- 0.6 fmol; the average percentage of serotonin released was 28 +/- 14%. Events that are consistent with released serotonin from single submicrometer granules (250 aL each) were evident, each of which contained an average amount of 5.9 +/- 3 amol.

Mechanisms of vancomycin-induced histamine release from rat peritoneal mast cells.

The mechanisms of vancomycin (VCM)-induced histamine release were studied with rat peritoneal mast cells. VCM (> 1 x 10(-3) M) released histamine from the isolated mast cells in a dose-dependent and noncytotoxic manner. In the absence of extracellular Ca2+, the histamine release was reduced markedly. When the intracellular Ca2+ was depleted, it was further decreased. The Fura-2-loaded single mast cells showed a biphasic increase in the intracellular Ca2+ concentration ([Ca2+]i) by VCM: the first transient and the second sustained components. In the absence of extracellular Ca2+, the transient component was unchanged, while the sustained component was eliminated completely. The IP3 content in the mast cells increased within 10 s after the application of VCM. These results suggest that VCM release histamine from rat peritoneal mast cells via an IP3 production and increase in [Ca2+]i.

Determination of the number of mast cells in lymph node, bone marrow, and buffy coat cytologic specimens from dogs

Summary Cytologic samples of popliteal lymph node, proximal femoral bone marrow, and the buffy coat fraction of blood were obtained from 56 dogs. The number of mast cells on 1 slide of each sample was determined by microscopic examination. Eleven of 46 slides of lymph node aspirate contained mast cells (range, 1 to 16; mean, 6.4; median, 5 mast cells/slide). Fifty-one bone marrow aspirate slides were evaluated. Two of these contained a single mast cell. None of the 53 buffy coat smear slides examined contained any mast cells. These results indicated that in clinically normal dogs, a few to several mast cells may be encountered in smears of lymph node aspirate, mast cells are rare in smears of bone marrow aspirate, and mast cells are absent from smears of buffy coat.

Monitoring exocytosis from single mast cells by fast voltammetry

We have used fast differential ramp voltammetry with carbon-fibre electrodes to monitor exocytotic secretion in single rat mast cells. The oxidation peak and other aspects of the electrochemical profile of the substance released were similar to those of 5-hydroxytryptamine (5-HT) and the signals were increased by preloading the secretory granules with exogenous 5-HT. Metabolic blockade inhibited both visible degranulation and the electrochemical signal. For comparison, quinacrine, which is fluorescent and accumulates in secretory vesicles, was used as an alternative means of detecting secretion in single cells. The amplitude of the electrochemical signals observed during degranulation correlated well with the loss of quinacrine fluorescence. Both methods were used to record successive rounds of secretion in single mast cells in response to repeated applications of compound 48/80.

A novel source of mast cells: The human placenta

The presence of moderate amounts of histamine in the human placenta was confirmed (0.72 +/- 0.10 microgram/g wet weight), and the hitherto unknown storage site of this biogenic amine was elucidated. Mast cells were identified by their characteristic morphology, staining reactions and secretory activity measured in terms of histamine release. Human placental tissue contains 7.6 x 10(5) mast cells/g wet weight, identified by staining with toluidine blue or alcian blue, and these cells were positive for chloro-acetate-esterase. Light microscope studies of placental tissue stained with HRP-conjugated anti-human IgE demonstrated cells with a typical 'halo' effect indicating cell-bound IgE, and electron microscopy revealed cells containing membrane-bound electron dense granules. A single mast cell was calculated to contain approximately 1 pg of histamine. Enzymatic digestion of placental tissue with collagenase (1.5 mg/ml) yielded viable cell suspension. containing mast cells in a purity of 0.6% which exhibited a low spontaneous output of histamine (12%). Placental mast cells released histamine in a concentration dependent manner upon challenge with anti-human IgE and the calcium ionophore A23187. Also, unlike other human mast cells so far studied. with the exception of skin, those dispersed from human placenta were responsive to the polybasic secretagogue compound 48/80. These findings represent a novel source of human mast cells and, since placentas are readily available in quantity, such tissue is proposed as an ideal source of mast cells for biochemical and pharmacological use.

Final steps in exocytosis observed in a cell with giant secretory granules.

Secretion by single mast cells was studied in normal and beige mice, a mutant with grossly enlarged secretory vesicles or granules. During degranulation, the membrane capacitance increased in steps, as single secretory vesicles fused with the cell membrane. The average step size was 10 times larger in beige than in normal mice, in agreement with the different granule sizes measured microscopically in the two preparations. Following individual capacitance steps in beige mice, individual granules of the appropriate size were observed to swell rapidly. Capacitance steps are frequently followed by the stepwise loss of a fluorescent dye loaded into the vesicles. Stepwise capacitance increases were occasionally intermittent before they became permanent, indicating the existence of an early, reversible, and incomplete state of vesicle fusion. During such "capacitance flicker," loss of fluorescent dye from vesicles did not occur, suggesting that the earliest aqueous connection between vesicle interior and cell exterior is a narrow channel. Our results support the view that the reversible formation of such a channel, which we term the fusion pore, is an early step in exocytosis.

An ultrastructural study of mast cells in the alveolar wall of normal and asthmatic lung.

Specimens of normal and asthmatic lungs were studied at the electron microscopic level and the frequency and ultrastructural features of mast cells and their granules within the alveolar wall were assessed with morphometric techniques. The numerical density of mast cells per square millimetre of alveolar wall was 299 (SD = 258) in normal and 366 (SD = 260) in asthmatic lung. The mean area of the mast cell nucleated profile was 25.7 microns2 (SD = 6.3) in normal lung and 29.8 (SD = 6.2) in asthmatic. The average number of secretory granules per single mast cell nucleated profile was 55 (SD = 13) in normal lung and 60 (SD = 12) in asthmatic lung. The diameter of the individual secretory granule was 338.9 nm (SD = 42.6) in normal and 345.6 (SD = 47.7) in asthmatic lung. The volume density of secretory granules in normal and asthmatic lung was 6.31 microns3 and 5.81 microns3, respectively. The mean diameter of the individual subunit ('scroll') inside the secretory granule was 88.8 nm for both normal and asthmatic lung. In normal lung 64.2% of granules were of 'scroll' and 'combined' type, and 35.8% of granules were 'particulate' or 'empty'. In specimens from asthmatic patients 40.3% of granules had 'scroll' or 'combined' structures and 59.7% were 'particulate' or 'empty'. Our data suggest that there is no difference between the number of mast cells in normal and asthmatic lung. However, in pulmonary mast cells from asthmatic lung, degranulation is more common than in normal lung.

Intraepithelial migration of mucosal mast cells in hay fever: ultrastructural observations.

Evidence has been presented suggesting that a migration of nasal mast cells from the mucosal connective tissue stroma into the epithelium is part of the mucosal response in birch pollen allergy. In a previous study, the identification of these intraepithelial cells as tissue mast cells rather than blood basophils was based on light microscopical morphology and histochemistry. We have now studied the ultrastructure of these cells in mucosal biopsies taken before and during the birch pollen season. Intraepithelial cells with basophil or metachromatic granules were only observed in biopsies taken during the season. Some of these cells had the ultrastructural appearance of tissue mast cells, including cytoplasmic lipid droplets and a granular substructure composed of multilamellar arrays and scrolls, serving to distinguish human mast cells from blood basophils. The ultrastructural traits of the remaining cells were heterogeneous, some reminiscent of human blood basophils, others of globule leucocytes of other species, but entirely typical blood basophils could not be identified. The results thus support our previous suggestion that a migration of mucosal mast cells from the connective tissue stroma into the epithelium is part of the human allergic mucosal response. It cannot be determined whether the ultrastructural heterogeneity of these cells is the result of an adaptation to the intraepithelial environment of one single mast cell type or to the existence of an ultrastructurally distinct mucosal mast cell.

Homology of the rat basophilic leukemia cell and the rat mucosal mast cell.

Secretory granules of the rat basophilic leukemia (RBL-1) cell, a chemically generated tumor cell line maintained in tissue culture, were shown to stain with alcian blue but not with safranin counterstain and to have sparse, small, electron-dense granules. A Mr 25,000 protein was the major [3H]diisopropyl fluorophosphate-binding protein in extracts of RBL-1 cells. Double-immunodiffusion analysis of extracts revealed immunoreactivity for rat mast cell protease (RMCP)-II, a Mr 25,000 neutral protease present in the secretory granules of rat mucosal mast cells and cultured rat bone marrow-derived mast cells, but no immunoreactivity for RMCP-I, the predominant neutral protease of rat connective tissue mast cells. By radial immunodiffusion, there was 66.8 ng of RMCP-II per 10(6) cells. Whereas rat connective tissue mast cells stain with alcian blue and safranin and contain heparin proteoglycan, rat mucosal and rat bone marrow-derived mast cells stain with alcian blue only and contain a non-heparin proteoglycan and lesser amounts of histamine. Proliferation of rat mucosal mast cells in vivo and rat bone marrow-derived mast cells in vitro requires T-cell factors, whereas no comparable requirement has been observed for connective tissue mast cells. The transformed RBL-1 tumor cells, whose growth is independent of factors other than those present in standard tissue culture medium, has previously been shown to contain predominantly chondroitin sulfate di-B proteoglycans and low amounts of histamine. The similar histology and secretory granule biochemistry of the rat mucosal mast cells, rat culture-derived mast cell, and RBL-1 cell suggest that they comprise a single mast cell subclass distinct from the rat connective tissue mast cell.

Proliferation of peritoneal mast cells in the skin of W/Wv mice that genetically lack mast cells.

Presence of mast cell precursors in the mouse peritoneal cavity was demonstrated, and the precursors were characterized. When a cell suspension, containing mast cell precursor(s), was directly injected into the skin of genetically mast cell-deficient WBB6F1 (WB X C57BL/6)-W/Wv mice, a cluster composed of approximately 2,000 mast cells appeared at the injection site. By determining the proportion of injection sites at which the mast cell cluster appeared, the concentration of mast cell precursors can be calculated by limiting dilution analysis. The concentration in the peritoneal cavity was about five times as great as the concentration in the bone marrow. Although peritoneal mast cell precursors were shown to originate from the bone marrow, physical characterization revealed that the peritoneal precursors differed from the marrow precursors. The peritoneal precursors were less susceptible to irradiation than the marrow precursors; the former were heavier than the latter. When a 95% pure mast cell suspension was prepared from the peritoneal cells by the removal of phagocytes and the density gradient centrifugation, 1 out of 16 cells had the potentiality to make a mast cell cluster in the skin of the W/Wv mice. Moreover, when a single mast cell was identified under the phase contrast microscope and picked up with the micromanipulator, 1 out of 17 mast cells made the cluster. This indicated that some peritoneal mast cells kept extensive proliferative potentiality even after morphological differentiation. In other words, some peritoneal mast cells themselves may function as the committed precursors.

Phosphorylation of a single mast cell protein in response to drugs that inhibit secretion

Phosphorylation of a single mast cell protein in response to drugs that inhibit secretion

Interaction of phosphatidylserine with mast cells.

Phosphatidylserine (PtdSer) potentiates histamine secretion from mast cells exposed to concanavalin A and Ca2+. In order to identify the form of PtdSer that is responsible for its effect on mast cell secretion, PtdSer containing a tritium-labeled serine moiety (3H-PtdSer) was synthesized from egg yolk phosphatidylcholine. The critical micelle concentration (CMC) of 3H-PtdSer and the binding isotherm for 3H-PtdSer interaction with mast cells were determined. The midpoints of the binding isotherm and the dose-response curve for potentiation of secretion coincide and are 2 orders of magnitude greater than the CMC. The shape of the binding curve is explicable either in terms of simple binding of preformed PtdSer micelles or of cooperative binding of monomeric PtdSer in which the number of molecules cooperatively associating with a mast cell binding site is equal to the number of monomers in a PtdSer micelle. In either case, at equilibrium, PtdSer micelles are bound to the mast cells. The number of PtdSer molecules bound to a single mast cell at equilibrium was estimated to be 3.7 X 10(9).

H-2 antigens, on mast cell membrane, as target antigens for anaphylactic degranulation

When single mast cells were isolated by micromanipulation, specific H-2 antigen-bearing mast cells were degranulated upon incubation with alloimmune sera (DAAD). When specific alloantigens were presented by lymphoid cells only, no degranulation occurred. Only antigen-bearing mast cells were degranulated, irrespectively of the presence of antigen-bearing lymphoid cells. Therefore, in DAAD, anaphylactic alloantibodies can and must recognize specific H-2 antigens on the mast cell membrane and simultaneously deliver the degranulation signal, through an Fc-Fc receptor interaction on the surface of the same mast cell.

Classical and alloimmune anaphylactic degranulation of isolated single mast cells

Viable mast cells, directly isolated by micromanipulation from a mouse peritoneal cell suspension, were deposited on the bottom of microtiter-plate wells and submitted to histamine release. Conventional antigen-induced anaphylactic degranulation as well as direct allogeneic anaphylactic degranulation were strongly inhibited when these mast cells were settled on normal tissue culture plastic surfaces. Nevertheless, normal degranulation could be recovered by pretreatment of the experimental surface with a multipositive charged molecule (poly-L-lysine).Under these conditions, we demonstrate that the degranulation of one isolated mast cell is possible and consequently, as regards the direct allogeneic anaphylactic degranulation, confirm the “self-triggering mechanism” in which the recognition of histocompatibility antigens on the membrane of the mast cell itself is the trigger to the secretory response.The technique of monocellular degranulation described in this paper provides a new tool which leads us to think that the problem of detection of anaphylactic antibody-secreting cells can be solved.

Zur Frage der Brauchbarkeit isolierter Mastzellen aus Adenoiden zur Diagnose von Inhalationsallergien

The enzymic tissue dispersion method yields single mast cells from human adenoids. These cells are light- and electronmicroscopically intact. The mast cells react to the calcium-ionophor A 23187 and to anti-human-immunglobulin E or acetylcholine after passive sensitization with histamine release. After passive sensitization with serum of patients suffering from hay feaver the specific antigen induces a histamine release.

Epidermal Mast Cells

Tissue from 134 patients with neurodermatitis and prurigo, pseudoepitheliomatous hyperplasia, pemphigus, and urticaria pigmentosa was examined qualitatively for epidermal mast cells. Epidermal mast cells were found in all of the diseases that were studied except dermatitis herpetiformis. Pemphigus vegetans and dermatitis vegetans were frequently associated with the presence of epidermal mast cells. In other pseudoepitheliomatous diseases, such as tuberculosis verrucosa cutis, blastomycosis, and bromoderma, epidermal mast cells were present in many cases. Four of eight patients with acral dermatitis with elevated IgE blood levels had intraepidermal mast cells; the number was much lower in patients with usual neurodermatitis and prurigo nodularis. Only two of ten cases of alopecia mucinosa showed epidermal mast cells. A single epidermal mast cell was found in ten cases of urticaria pigmentosa. Chronic inflammation associated with epidermal cell proliferation appeared to correlate with the presence of epidermal mast cell.

Histamine Release from Isolated Rat Mast Cells in Metal Ion-Free Medium*

It is generally recognized that the histamine release from mast cells induced by a number of histamine releasers including compound 48/80 or by antigen-antibody reaction is accompanied by extracellular discharge of granules (degranulation), though this is not always the case (1–3). Uvnäs and Thon (4, 5) proposed a hypothesis that the mechanism of histamine release from mast cells by compound 48/80 consists of two consecutive steps: the one being the process of extracellular discharge of granules, and the other, that occurs subsequently, a process of the release of histamine from extracellular granules. They consider that the first process is energy-requiring but the second one is a non-enzymatic simple ion exchange which occurs in the extracellular fluid phase between granular histamine and inorganic cations. This hypothesis is largely based on the following observations: 1) Mast cells which have degranulated in vitro on exposure to compound 48/80 retain their ability to store granules and histamine, and also to discharge granules and histamine on a second exposure to compound 48/80. 2) In an isotonic sucrose solution, granules are discharged from mast cells by compound 48/80 but histamine is not released; if these granules are resuspended in a medium containing inorganic cations, histamine release does occur. The first observation seems to be substantiated by recent findings of Diamant et al. (6) and Tasaka et al. (7) that when compound 48/80 is repeatedly applied topically to the surface of a single mast cell by means of microelectrophoresis, degranulation occurs in response to each application. We have failed to wholly reconfirm the latter observation, however, since in our experiment, histamine release from mast cells was produced to a considerable degree by compound 48/80 in isotonic sucrose solution, although an additional histamine release could be observed from mast cells resuspended in NaCl solution.

Histamine release from isolated rat mast cells in metal ion-free medium.

It is generally recognized that the histamine release from mast cells induced by a number of histamine releasers including compound 48/80 or by antigen-antibody reaction is accompanied by extracellular discharge of granules (degranulation), though this is not always the case (1-3). Uvnas and Thon (4, 5) proposed a hypothesis that the mechanism of histamine release from mast cells by compound 48/80 consists of two consecutive steps: the one being the process of extracellular discharge of granules, and the other, that occurs subsequently, a process of the release of histamine from extracellular granules. They consider that the first process is energy-requiring but the second one is a non-enzymatic simple ion exchange which occurs in the extracellular fluid phase between granular histamine and inorganic cations. This hypothesis is largely based on the following observations: 1) Mast cells which have degranulated in vitro on exposure to compound 48/80 retain their ability to store granules and histamine, and also to discharge granules and histamine on a second exposure to compound 48/80. 2) In an isotonic sucrose solution, granules are discharged from mast cells by compound 48/80 but histamine is not released; if these granules are resuspended in a medium containing inorganic cations, histamine release does occur. The first observation seems to be substantiated by recent findings of Diamant et al. (6) and Tasaka et al. (7) that when compound 48/80 is repeatedly applied topically to the surface of a single mast cell by means of microelectrophoresis, degranulation occurs in response to each application. We have failed to wholly reconfirm the latter observation, however, since in our experiment, histamine release from mast cells was produced to a considerable degree by compound 48/80 in isotonic sucrose solution, although an additional histamine release could be observed from mast cells resuspended in NaCl solution. The present paper offers evidence of such a release of histamine induced by compound 48/80 in isotonic sucrose solution free of metal ions and describes some observations made for elucidation of the mechanism of this phenomenon. A short account of this work has been published (8).

A Fluorescent Study of Histamine Release from a Single Mast Cell of the Rat Mesentery

A Fluorescent Study of Histamine Release from a Single Mast Cell of the Rat Mesentery