ARTICLE |
Correspondence to: Gary R. Login, Dept. of Pathology, Beth Israel Hospital, 330 Brookline Ave., Boston, MA 02215.
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Summary |
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The subcellular events responsible for release of mediators by mast cells may help to clarify roles for mast cells in health and disease. In this study we show that the granule-associated protease chymase is also within cytoplasmic vesicles in appropriately stimulated rat peritoneal mast cells. Rat peritoneal mast cells were recovered before or 1-10 sec after exposure to the secretogogue compound 48/80 (10 µg/ml) and then were examined by radioimmunoassay to quantify histamine release or were processed, using routine methods for postembedding immunoelectron microscopy, to identify the subcellular localization of chymase. In comparison to unstimulated cells, compound 48/80 stimulated cells in two independent experiments showed an increase (15%, 28%) in the surface area of the cell and a decrease (12%, 6%) in the surface area of the total granule compartment before degranulation channel formation. These global cellular changes occurred in a background of transient but significant (p<0.01) increases in the area and number of chymase-immunoreactive vesicles per µm2 cytoplasm. These changes were detectable at 5 or 7 sec after stimulation with compound 48/80 but returned to near prestimulation levels by 9 or 10 sec after addition of compound 48/80 (total cumulative histamine release was 28% by 8 sec and 47% by 14 sec). These observations suggest that vesicles participate in the early stages of regulated secretion of chymase from rat peritoneal mast cells. (J Histochem Cytochem 45:1379-1391, 1997)
Key Words: rat, vesicular transport, secretion, morphometry, electron microscopy, immunocytochemistry, mast cell, chymase
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Introduction |
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Mast cells can be activated to undergo degranulation, resulting in the release of histamine, proteases, and other cytoplasmic granule-associated mediators, in response to a wide variety of stimuli, including IgE and specific antigen, complement-derived peptides, and basic compounds, including compound 48/80 (
Most biochemical and morphological analyses of mast cell mediator release have focused on the process of anaphylactic degranulation, also designated compound exocytosis. This process is characterized morphologically by granule swelling followed by the relatively rapid fusion of the membranes surrounding individual cytoplasmic granules with each other and with the plasma membrane (
Ultrastructural analyses of both basophils (
Certain aspects of this hypothesis have been supported by morphological studies in basophils (
In this study, we analyzed the subcellular localization of the cytoplasmic granule-associated protease chymase in compound 48/80-stimulated rat peritoneal mast cells, using postembedding immunocytochemical methods. We found that cytoplasmic vesicles that exhibit immunoreactivity for chymase increased in number and area fraction during a brief period of time (5 or 7 sec) after stimulation of the cells with compound 48/80. These chymase-immunoreactive vesicles then decreased in number, in concert with the formation of degranulation channels. Although published reports have examined chymase localization in storage granules (
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Materials and Methods |
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Animals
Male Sprague-Dawley rats (virus-free) (Taconic Laboratories; Germantown, NY) weighing 250-350 g were used. These experiments were approved by the Beth Israel-Deaconess' Institutional Animal Care and Use Committee. The animal care program at the Beth Israel Hospital is accredited by the American Association for the Accreditation of Laboratory Animal Care and meets National Institutes of Health standards as set forth in the "Guide for the care and use of laboratory animals" [DHHS publication (NIH) 86-25, revised 1985]. The animals were housed in the Beth Israel Hospital's animal care facility, where they were provided with rat chow and water ad libitum and were allowed to acclimate for approximately 2 days before mast cell harvest.
Preparation and Stimulation of Peritoneal Mast Cell Suspensions
Rat peritoneal mast cells were washed from the peritoneal cavities of rats with Hank's Balanced Salt Solution (HBSS) (
In our standard experiment, cells at 4C [note: reaction temperatures below 17C inhibit exocytosis (
Rapid Collection and Determination of Released Histamine
Mast cells (from an aliquot distinct from that used for the immunocytochemical studies) were stimulated at RT by injecting a 0.3-ml cell aliquot (1.4 x 105 cells) into a 2.7-ml volume of HBSS containing compound 48/80 through a port in a 12-ml syringe (Monoject; St Louis, MO) that was connected to a 0.5-µm Millipore filter (Millipore; Bedford, MA). Aliquots of supernatant (0.37 ml) were collected at intervals of 2 sec by pushing the syringe plunger. Histamine was measured using a radioimmunoassay kit (Immunotech; Westbrook, ME). The normalized histamine release (i.e., the pg/ml released during a specific 2-sec interval minus the spontaneous release at 0 sec) and the percent total (cumulative) histamine release at each time point were computed by taking into account the changes in supernatant volume that occurred over the course of the experiment. In this experiment, mast cells contained 30 pg of histamine/cell.
Fixation, Processing, and Embedding for Light and Electron Microscopy
Mast cells in HBSS (total volume 1 ml) were added to 14 ml of Karnovsky's fixative (2% formaldehyde, freshly prepared from paraformaldehyde, 2.5% glutaraldehyde, 0.025% CaCl2, 0.1 M sodium cacodylate buffer, pH 7.4) for 1 hr at 20°C. After fixation in suspension, cells were pelleted at 800 x g for 10 min, resuspended in 10 ml of 0.1 M sodium cacodylate buffer, pH 7.4, 4C, and washed in sodium cacodylate buffer two times at 800 x g for 5 min at 4C.
Cell suspensions were pelleted through molten agar (
Immunocytochemistry of Chymase
Postembedding immunogold labeling of chymase on Epon sections was done as follows (
Controls included substitution of normal goat serum from a nonimmunized animal to evaluate the specificity of the primary antibody and omission of the primary antibody to evaluate nonspecific binding of the secondary gold-labeled antibody. Appropriate absorption controls for the primary antibody for rat mast cell granule chymase have been reported (
Quantitative Analysis of Mast Cell Stimulation
Cell blocks from each experimental condition, in experiments performed with cells from three different groups of animals, were selected at random. Sampling procedures were done in accordance with procedures of stratified random sampling (5% of the granules per 1-µm-thick mast cell section), and extensively stimulated ("extensively degranulated") (i.e., <10 blue-stained granules remaining in the cytoplasm per 1-µm-thick mast cell section). The mean and SD of the number of unstimulated, minimally to moderately stimulated, and extensively stimulated cells were calculated for each time point.
Quantitative Analysis of Gold-labeled Vesicles (GLVs) (Indicating Chymase Immunoreactivity)
Thin sections were examined systematically in the electron microscope in the same order, beginning at the top left corner of the grid and proceeding to the lower right corner of the grid, until at least five mast cells were photographed. Electron micrographs of complete cell profiles were printed at x13,750 (21.5 x 25.5 cm). Electron micrographs of immunogold-labeled sections were printed at x41,250 and five prints containing a combined minimum of 50 granules from each experimental and control group were used for stereological analysis. Cell images included the nucleus, plasma membrane, multiple cytoplasmic granules, and mitochondria. Cytoplasmic vesicles were identified according to the following criteria (
Area determinations were done by a point-counting method using a square lattice overlay (
Quantitative Analysis of the Surface Areas of Granule and Cytoplasmic Membranes in Unstimulated and Compound 48/80-stimulated Mast Cells
Established point-counting methods were used (
The volume fraction (Vv) of granules (g) was determined from their areas (A). The numerical granule density (Nvg) per µm3 of cytoplasmic volume was determined by
Nvg = Vvg/g (
where the mean granule volume (g) was calculated by using the formula:
g = ß' (
g)3/2 (
as modified for use with mean particle volumes (n) was derived according to:
For determination of granule number and volume per cell, we first determined the nuclear volume fraction (vn) by using profiles of whole cells as the reference. The cell volume was calculated according to
n/
vn. We then calculated the total number of granules/cell (Ng) according to:
Ng = Nvg (cell -
n) (
Surface area to volume ratio of the cell (Svcell), µm2 of plasma membrane per µm3 of cell volume, was determined by counting the number of horizontal and vertical test lines on a screen crossing the cell perimeter and by dividing by the number of intersections falling on the cell. This ratio was multiplied by a correction factor for screen size, microscope magnification, and print magnification. Surface area to volume ratio of the granule, Svg, µm2 of granule membrane per µm3 of granule volume, was calculated by the same procedure using a screen size appropriate for granules. The surface area (SA) of the cell was determined by the product of Svcell and cell. The surface area of the total granule compartment was determined by the product of Svg,
g, and Ng.
Statistical Analysis for Light Microscopy and Immunocytochemical Studies
Differences among conditions were examined for statistical significance using one-way, unblocked ANOVA. Pair-wise differences were evaluated by the Newman-Keuls multiple sample comparisons test. p<0.05 was considered a significant difference. Data organization and analysis were performed on the PROPHET system, a national computer resource sponsored by The National Center For Research Resources, National Institutes of Health.
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Results |
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Rat Peritoneal Mast Cells Exhibit a Time-Dependent Degranulation Response to Stimulation by Compound 48/80
Measurements of the percent of total (cumulative) normalized histamine release showed that unstimulated mast cells (Figure 1A time) exhibited only 1% spontaneous histamine release, whereas stimulation of the mast cells with 10 µg/ml compound 48/80 at 20C for 14 sec induced the release of 47% of the cells' histamine (Figure 1A). This latter finding is consistent with the literature (
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A progressive increase in the mean percentage of mast cells that exhibited light microscopic evidence of activation (defined as cells whose cross-section contained >5 pink-stained, swollen cytoplasmic granules) occurred between 6 sec (40 ± 10%) and 10 sec (88 ± 8%) after stimulation with compound 48/80 (Figure 1B). The percentage of cells showing morphological evidence of extensive degranulation (i.e., <10 blue-stained granules remaining in the cytoplasm per 1-µm-thick mast cell section) was 12% by 9 sec and 33% by 10 sec (Figure 1B). There was no significant difference between the mean percentage of cells showing features of partial degranulation between 1 and 5 sec after addition of compound 48/80 (data not shown).
By light microscopy of alkaline Giemsa-stained, Epon-embedded 1-µm sections, most of the cytoplasmic granules in unstimulated mast cells stained dark blue and were densely packed in the cytoplasm. However, a few granules appeared somewhat larger than these typical blue-stained granules, and stained pink. We chose five as the maximal number of such pink granules that could be exhibited in a single 1-µm-thick section of a "non-degranulated" mast cell. We designated those mast cells with six or more such granules (but with 10 dense, blue-stained granules) as "partially degranulated." However, low content of histamine in the medium of unstimulated mast cells (Figure 1A time) suggests that the presence of small numbers of altered, pink-staining cytoplasmic granules in the mast cells that had not been stimulated with compound 48/80 represents a "baseline" finding of changes occurring either in vivo or during the purification of the cells.
At 6 sec after mast cells were incubated with compound 48/80, there was a progressive increase in the number and size of pink-stained granules. By 9 sec after addition of compound 48/80, large pink-staining, irregularly shaped areas dominated the cytoplasm; cells also retained individual dark blue- or pink-stained granules. Consistent with published reports (
Ultrastructural Features of Mast Cells During the First 10 Seconds After Exposure to Compound 48/80
Many of the ultrastructural features of anaphylactic-type degranulation in rat peritoneal mast cells have been described in detail (
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Stimulated cells that were observed 7-8 sec after the addition of compound 48/80 exhibited some altered granules (
By 7-8 sec after addition of compound 48/80, vesicles with smoothly contoured, circular, or elliptical profiles, often with electron-dense contents, and diameters between 50 and 200 nm were a dominant feature in the cytoplasm and often occurred close to or in contact with the granule membrane (Figure 2B). Vesicles were observed around mature cytoplasmic granules regardless of the proximity of the granules to the plasma membrane. Cells that had been stimulated with compound 48/80 for 9 sec exhibited degranulation channels containing extruded, swollen, membrane-free altered granule matrices. Extrusion of membrane-free, nonmembrane-bound granules to the exterior of the cell was also apparent. Few electron-dense granules remained in the stimulated cells, and these granules typically were clustered around the nucleus.
Immunolocalization of Chymase to Cytoplasmic Granules in Mast Cells After Stimulation with Compound 48/80
Immunolocalization of chymase, a major constituent of the granules of rat peritoneal mast cells (
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Stimulation of Mast Cells with Compound 48/80 Is Associated with a Rapid but Transient Increase in Chymase-immunoreactive Cytoplasmic Vesicles
We determined the area (µm2) and number of chymase-immunoreactive vesicles and total vesicles per µm2 cytoplasm (area fraction and number fraction, respectively), as well as the gold labeling density in all vesicles, in unstimulated or compound 48/80-stimulated mast cells (Table 1). In Exp. 1 we established that changing unstimulated mast cells from 4C to RT for 7 sec (without also adding compound 48/80) resulted in no statistically significant differences in the numbers of cytoplasmic vesicles. However, compared to the values in unstimulated cells, mast cells stimulated with compound 48/80 for 5 sec exhibited a 271% increase in the area fraction of gold-labeled vesicles (5 sec, 0.0052 µm2 GLVs/µm2 cytoplasm vs 0 sec, 0.0014 µm2 GLVs/µm2 cytoplasm; p<0.01), a 450% increase in the number of gold-labeled vesicles (5 sec, 0.77 GLVs/µm2 cytoplasm vs 0 sec, 0.14 GLVs/µm2 cytoplasm; p<0.01), and a 110% increase in the gold label density in the entire vesicle compartment (5 sec, 78 gold particles/µm2 total vesicles vs 0 sec, 37 gold particles/µm2 total vesicles; p<0.01), as well as a small (43%) but not significant decrease in the average size of the gold-labeled vesicles (Exp. 2, Table 1). By contrast, cells stimulated for 10 sec, in which well-developed degranulation channels were evident, showed no significant differences in any of these values compared to those in unstimulated cells or in cells that had been stimulated for only 1 second (Table 1). Similar to the compartment of gold-labeled vesicles but less dramatic were changes in the total vesicle compartment with respect to measurements of the area fraction and number of vesicles/µm2. However, there were no significant differences with respect to the average vesicle size (µm2) (Table 1).
In Exp. 3, the changes in the gold-labeled vesicle compartment, as assessed by comparing results from unstimulated mast cells and cells examined 7 sec after exposure to compound 48/80, were similar to those in the Exp. 2. There were significant (p<0.01) increases of 237% in the area of GLVs/µm2 cytoplasm, of 280% in the number of GLVs/µm2 cytoplasm, and of 277% in the gold density in all vesicles (Table 1; Figure 1, Figure 4, and Figure 5). By 9 sec after stimulation with compound 48/80, each of these values had returned to levels that were not significantly different from the corresponding values in unstimulated cells (Table 1; Figure 1). Although there were no significant differences in the area fraction for the total vesicle compartment between unstimulated cells and cells stimulated with compound 48/80, the average size of individual vesicles in the total vesicle compartment by 9 sec after stimulation showed a small (40%) but significant (p<0.01) increase and the number of vesicles by 9 sec showed a small (60%) but significant decrease (p<0.01) compared to unstimulated cells (Table 1).
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Our data indicate that GLVs did not differ significantly from unlabeled vesicles in average size (Table 1), nor was the dramatic increase in area fraction or number of GLVs in these cells reflected in changes in these values for the total vesicle compartment. Indeed, the area and number of GLVs/µm2 cytoplasm never accounted for more than 35% and 41%, respectively, of the total vesicle compartment in Exp. 2, or for more than 27% and 31%, respectively, of the total vesicle compartment in Exp. 3. We also found that the density of gold label in the total vesicle compartment was highest when the number of gold-labeled vesicles peaked in both Exp. 2 (at 5 sec) and Exp. 3 (at 7 sec), and the differences between these values and those in unstimulated cells were statistically significant (p<0.01, p<0.05, respectively).
Compound 48/80-stimulated Mast Cells Show an Increase in the Surface Area of the Cell and a Decrease in the Surface Area of the Total Granule Compartment in the Time Preceding Exocytosis
We determined the surface area of the cell and of the total granule compartment in unstimulated or compound 48/80-stimulated mast cells (Table 2). The surface area of mast cells increased by 15% and the surface area of the total granule compartment decreased by 12% after stimulation for 5 sec. In a second experiment, the surface area of mast cells increased by 28% and the surface area of the total granule compartment decreased by 6% after stimulation by compound 48/80 for 7 sec.
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A Model Illustrating the Possible Fates of Chymase-immunoreactive Vesicles During Mast Cell Degranulation
Most unstimulated mast cells (Figure 2A, Figure 5A, and 6) contained granules with closely approximated, smoothly contoured membranes. As reported by
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Discussion |
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In this study, we present the first immunocytochemical evidence that vesicles that contain granule-associated chymase increase in number and area fraction at very early intervals (5-7 sec) after exposure of rat mast cells to compound 48/80. By using a rapid cell handling approach with 1-sec resolution, we show that chymase-labeled vesicle activity precedes degranulation channel formation in these cells and is associated with a significant reduction in the density of chymase-immunoreactivity in altered cytoplasmic granules that formed during the secretory process. Our approach further shows the feasibility of studying exocytotic processes in intact mast cells, with a time resolution comparable to those reported in recent studies for permeabilized mast cells (
We also used morphological, morphometric, and biochemical methods on replicate samples of similarly rapidly handled cells and supernatants to establish the time-line between pre-exocytotic morphological changes in granules and vesicles and the parallel release of the granule mediator histamine. Importantly, in rat peritoneal mast cells compound 48/80-induced secretion of histamine and activation of chymase are initiated by the same serine protease reaction (
Our findings show that compound 48/80 stimulation is associated with a rapid, transient increase in the chymase-immunoreactive vesicle compartment and, to a lesser extent, in the total vesicle compartment in the post-stimulus interval up to 10 sec. This change coincided with the appearance in these cells of altered granules with diminished chymase immunoreactivity and immediately preceded the formation of degranulation channels. In addition, this change coincided with a decrease in the surface area of the granule compartment and an increase in the surface area of the cell membrane. Interestingly, Kraeuter Kops et al. (1990) report a similar increase in the number of total vesicles in compound 48/80-stimulated rat peritoneal mast cells (0.9 vesicles/µm2 cytoplasm in unstimulated cells and 1.9 vesicles/µm2 cytoplasm in stimulated cells; stimulation time not reported).
The findings of vesicle-like structures in continuity with the membranes of mature cytoplasmic granules (
What is the specific fate of the chymase-immunoreactive vesicles in compound 48/80-stimulated mast cells? As proposed by us (
It is also possible, as proposed in our general model of basophil or mast cell secretion (
Yet another role of a vesicle-mediated secretory pathway in mast cells may be the selective transport of granule mediators. Rat peritoneal mast cells stimulated by the combination of compound 48/80 and the tricyclic antidepressant amitriptyline have been shown by an autoradiographic method to differentially release serotonin without histamine and without degranulation (
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Acknowledgments |
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Supported by NIH grants DE-10059 (GRL), AI-33372 (AMD), and AI-20487 (LBS); the Brazilian Council of Research, Grant #200902/91 from CNPq (LOL); and the Department of Dermatology, Nippon Medical School, Tokyo, Japan (MA).
We thank Dr Koji Yano, Department of Pathology, for doing the histamine assays, Ms Patricia Fox for assistance in thin sectioning for electron microscopy, and Dr Ilan Hammel, Sackler School of Medicine, Tel Aviv, Israel, for his advice in the morphometric analysis.
Received for publication May 13, 1997; accepted May 27, 1997.
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