A Study of Allergic Subjects during and out of the Pollen Season |
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Abstract |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The variability of serum and plasma levels of eosinophil granule proteins in different clinical conditions, interpreted as the result of different patterns of cytokine priming, suggests a selective mobilization of granule proteins. Inasmuch as piecemeal degranulation (PM) is the mechanism proposed for the differential release of eosinophil granule proteins, we decided to investigate whether blood eosinophils from allergic subjects show characteristics of PM during natural allergen challenge. Eosinophils from three birch-sensitive subjects were studied before and during the pollen season. Electron microscopy analysis showed that during the season, eosinophils presented morphologic features of PM. By immunogold labeling, eosinophil cationic protein (ECP) was detected not only in normal specific granules but also in the cytoplasm, in the vicinity of partially lucent specific granules. These results were confirmed by subcellular fractionation, where the amount of ECP associated with compartments containing small vesicles increased 2-fold during the pollen season. A study of the distribution of ECP, eosinophil peroxidase, and hexosaminidase in eosinophils of different densities showed that the profile of each of these proteins differed depending on cell density. All of these proteins decreased in the specific granule of hypodense cells and increased in other cell compartments. We conclude that allergen exposure causes PM of the peripheral blood eosinophils of allergic subjects, and that the density of these cells reflects the degree of degranulation. Our results provide novel information for the understanding of the selective mobilization of granule proteins into the circulation.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The specific granules of eosinophils are both storage and secretory organelles. Eosinophil mediators such as cytokines, lysosomal hydrolases, and granule cationic proteins, namely eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), eosinophil protein X (EPX), and major basic protein, are stored in the specific granules and secreted upon cell stimulation. Three mechanisms of protein release have been postulated: exocytosis, piecemeal degranulation (PM), and cell lysis, of which PM has been proposed to explain the selective release of specific granule proteins. According to this model, the content of specific granules is packed into small vesicles and then transported to the cell membrane, leaving the specific granules gradually empty. This mechanism was inferred from ultrastructural studies of eosinophils derived from mononuclear cells cultured in the presence of interleukin (IL)-5 (1), where specific granules showed losses of crystal core and/or matrix to different extents, and vesicles of different shapes containing EPO were present in the vicinity of partially emptied specific granules. Similar findings have been reported in mature eosinophils cultured with IL-5 for 7 d (2), eosinophils incubated with viruses (3), and eosinophil myelocytes inhibited in their development (4). The selective release of eosinophil proteins upon cell stimulation has also been reported in other experimental systems (5, 6). Using clinical models, Erjefält and colleagues (7) observed the presence of eosinophils showing features of PM in the mucosa of the upper and lower airways of allergic subjects.
Serum levels of eosinophil granule proteins, namely ECP, EPO, and EPX, are considered as an indication of the degree of disease activity during different clinical conditions. Elevated serum levels of these proteins (8), as well as increased numbers of hypodense eosinophils (9), have been reported to be associated with the degree of activity of allergic disease. In patients with atopic dermatitis (10), serum levels of ECP were shown to correlate with the number of circulating hypodense eosinophils and not with normodense cells. We have previously observed morphologic changes of specific granules resembling the early stages of PM in eosinophils, along with a concomitant increase in the serum levels of ECP, EPX, and EPO (11), in healthy subjects 24 h after administration of granulocyte colony-stimulating factor.
The aim of the present work was to study the occurrence of PM in peripheral blood eosinophils from allergic subjects before and during the pollen season, both by electron microscopy (EM) and subcellular fractionation techniques. We also investigated the relationship between eosinophil density and the protein content of different cell compartments. The conclusions reached contribute to the understanding of the selective mobilization of granule proteins in peripheral blood eosinophils.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Patients
Five allergic subjects and nine healthy volunteers were included in the study. The allergic subjects were all mild asthmatics treated with antihistaminic drugs. Blood samples from three of the allergic subjects were taken in February, before the birch pollen season, and in May, when the birch pollen count had reached 450 counts/m3. Separation of eosinophils of different densities was performed on cells obtained from the fourth nontreated, mildly allergic subject during the pollen season. Eosinophils from the fifth allergic subject were used for subcellular fractionation and further electron microscopic examination of cell organelles.
EM
The morphology of the organelles from one of the sucrose gradient fractionations of cells from allergic subjects during the pollen season was examined by EM. Fractions were pooled according to marker and eosinophil protein distribution profiles, as explained in Figure 4. The pools were gently diluted up to 8.5 mL with 10 mM [1,4-piperazine-bis ethane sulfonic acid] (Pipes) buffer, pH 7.3 (Sigma Chemical Co., St. Louis, MO), and left on ice for 1 h. Organelles were then fixed with 2.5% glutaraldehyde and 0.5% paraformaldehyde (final concentrations) in cacodylate buffer (pH 7.4) for 2 h on ice. The fixed organelles were pelleted by centrifugation at 25,000 rpm for 25 min at 4°C in a Beckman ultracentrifuge (SW 28.1 rotor). The pellets were washed with cacodylate buffer and postfixed with osmium tetroxide, dehydrated stepwise with ethanol (50, 75, 90, and 95% and absolute ethanol twice), and embedded in Spur's resin (Agar Scientific Ltd., Standstill, UK). Samples were sectioned with an Ultrotome V (LKB Bromma, Bromma, Sweden) provided with a diamond knife (Diatome, Bienne, Switzerland) and stained with uranyl acetate and lead citrate.
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Immunoelectron Microscopy
Blood for immunoelectron microscopy experiments was collected
in 4.5 mL ethylenediaminetetraacetic acid tubes (Vacutainer system; Becton Dickinson, Meylan Cedex, France). Leukocytes
were separated from erythrocytes by sedimentation on Ficoll Hypaque (Pharmacia Biotech AB, Uppsala, Sweden) for 30 min at
room temperature (RT). The leukocyte-containing plasma (upper
phase) was carefully collected and the leukocytes were washed
twice with phosphate-buffered saline (PBS) and fixed with 4%
paraformaldehyde plus 0.5% glutaraldehyde in cacodylate buffer
on ice for 2 h. The fixed cells were washed in Ringer's buffer, dehydrated in ethanol (50, 75, 90, and 95% and twice in absolute ethanol), and embedded in Lowicryl KM4 (Agar Aids). Polymerization was performed by ultraviolet light (360 nm) at 
20°C.
Sections of 50 nm were cut with an Ultrotome V provided with a
diamond knife. Sections were mounted on Formvar (Merck, Darmstadt, Germany)-coated golden grids.
Immunolabeling
The sections were first incubated in 0.1 M phosphate buffer (pH 7.4) with 0.15 M glycine, followed by incubation with 1% bovine serum albumin (BSA) in 0.1 M phosphate buffer (pH 7.4) to block unspecific binding. The sections were then incubated with primary antibody (EG2 mouse monoclonal antibody against ECP; Pharmacia & Upjohn Diagnostics, Uppsala, Sweden) at RT for 1 h, rinsed with 0.2% BSA in phosphate buffer, and incubated with secondary antibody (goat antimouse immunoglobulin [Ig] G) conjugated to 10 nm gold (British BioCell International, Cardiff, UK). The sections were finally washed and fixed with 3% glutaraldehyde in 0.1 M phosphate buffer. As controls we used samples which were incubated with secondary antibody only, or samples where the primary antibody was replaced by a nonrelevant antihuman murine IgG (Dakopatts, Glostrup, Denmark). Counterstaining of the sections was performed with 10% uranyl acetate in 100% methanol at 40°C. Samples were analyzed in a Hitachi 7100 (Hitachi, Tokyo, Japan) transmission electron microscope.
Subcellular Fractionations
Eosinophils were isolated from whole blood by negative magnetic immune selection as described by Hansel and associates
(12), with minor modifications. Red cells were hemolyzed twice
with water. Purified eosinophils were suspended in 1.5 mL of hypotonic sucrose (6% wt/wt sucrose in 10 mM Pipes, pH 7.3) and
left on ice for 15 min. Cell disruption was performed by sonication after comparison with other methods, namely nitrogen cavitation and repeated passage through a needle. Cells were sonicated for 15 s at an amplitude of 8 µm (Soni Prep sonicator;
Sanyo Gallenkamp, Leicester, UK) and the tonicity of the solution was restored by adding 0.185 mL of 34% (wt/wt) sucrose.
The sonication energy used was the lowest resulting in almost
100% disintegration, as estimated by observation in a Burk chamber after cell-staining with Turk dye and by evaluation of the
morphology of hematoxylin and eosin (H&E)-stained samples
by light microscopy. The use of twice the sonication energy or
twice the time length of sonication did not have any effect on the
ECP, EPO, and EPX subcellular distribution profiles obtained.
After sonication, intact cells and nuclei were spun down at
1,000 × g for 5 min at 4°C. The resulting postnuclear supernatant
was loaded on the top of a sucrose density gradient. Sucrose solutions (wt/wt) were made in 10 mM Pipes, pH 7.4. During the
course of this work we used two types of gradients. Gradient I
was partially continuous and consisted of a lower section made of
layers of 60, 55, 50, 46, and 43% sucrose that were left at RT for 3 h
before being overlaid with layers of 34, 32, 30, 25, and 20% sucrose. Postnuclear supernatants were loaded at the top of such
gradients within 10 min. Gradient II was totally continuous and
consisted of layers of 60, 55, 50, 46, 42, 38, 34, and 20% sucrose
left to diffuse for 3 h at RT before sample loading. After overnight centrifugation at 54,000 × g in a Beckman ultracentrifuge (SW28.1 rotor) at 4°C, 40 fractions of 425 µl each were collected from the top of the gradients by upward displacement with 60% sucrose. Samples were stored at 20°C until analyzed for enzyme activities and content of eosinophil proteins. Gradient I gave a
good resolution of the less-dense cell organelles (Golgi, plasma membranes, endocytic vesicles) whereas gradient II separated
better mitochondrial fractions and the different subpopulations
of specific granules from plasma membranes. The subcellular
fractionation methods used gave highly reproducible results. The
morphology of the organelles obtained using such gradients was
determined by EM as described earlier.
Separation of Eosinophils of Different Densities
Eosinophils of different densities were separated by Percoll gradient centrifugation according to Gärtner (13), with modifications. A total of 2 mL of a suspension of eosinophils (98% pure) in PBS containing 2% newborn calf serum was loaded on a Percoll gradient consisting of successively underlaid layers of freshly made Percoll solutions in PBS of the following densities (in g/mL): 1.075, 1.078, 1.086, 1.092, 1.100, and 1.111. Percoll solution densities were measured with a density meter (Paar, Graz, Austria). The gradient was centrifuged at 600 × g for 1 h at RT. The cells present as bands at the interfaces between Percoll layers were collected with a Pasteur pipette, washed twice with PBS, and subjected to subcellular fractionation as described earlier.
Marker Proteins
We used the following activities as organelle markers: 
-hexosaminidase (specific granules and lysosomes), 
-mannosidase II (Golgi apparatus and lysosomes [14]), and alkaline phosphodiesterase (PDE) I (plasma membranes). These activities were estimated as described by Storrie and Madden (15). Hexosaminidase
is widely considered to be a marker of lysosomes in a number of
cell types. In eosinophils it is also present in the specific granules
and can be released upon stimulation (16). In our gradients, we observed that some alkaline PDE and -mannosidase II are present in the specific granule compartment. The presence of markers for Golgi apparatus and plasma membranes associated with the specific granules (storage compartment) could be the consequence
of trafficking between compartments.
Albumin was used as marker for the subpopulation of vesicles containing CD11b receptors (17). The concentration of albumin was measured by radioimmunoassay using [I125]-labeled human serum albumin and polyclonal rabbit antibodies against human albumin (1:20,000 dilution). Albumin-antibody complexes were separated from unbound protein using the decanting suspension nb3 (Pharmacia & Upjohn Diagnostic). Determinations were performed in duplicate (coefficient of variation [CV] < 10%).
Total protein content was determined using a commercial kit (Bio-Rad, Hercules, CA).
ECP and EPO Assays
The ECP content of gradient fractions was measured by a specific radioimmunoassay (Pharmacia & Upjohn Diagnostics) (18). EPO was assayed by a specific fluoroimmunoassay as described elsewhere (19).
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Immunoelectron Microscopy
The morphologic appearance and ECP distribution of eosinophils from a nonsymptomatic allergic subject in February, before the pollen season, are illustrated in Figure 1. The majority of granules in most cells showed a crystal core and a dense, homogeneous matrix. Some specific granules showed signs of activation, such as a disintegrated or absent crystal core and/or a partially dissolved matrix. Labeling for ECP was mostly confined to the matrix of the specific granules. A weak labeling for ECP was also observed outside the granules, in the vicinity of the plasma membrane and in the central part of the cell. These ultrastructural features were similar to those of eosinophils from healthy controls (not shown).
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During the pollen season the morphology of eosinophils changed. Figure 2a shows an eosinophil from one of
the allergic subjects studied. Most of the specific granules
had an altered appearance: they lacked a crystal core and
the matrix compartment presented a loose structure. The
cytosol was often more electron-dense in the vicinity of
partially emptied granules than anywhere else (Figure 2b).
In other cells another type of alteration of the specific
granules prevailed
the crystal core was still present and
showed intact crystalline edges but a partly dissolved interior, while the matrix compartment appeared almost empty
(Figure 3). The cytoplasm in the vicinity of these partially
altered granules had the same electron density as elsewhere
in the cell. Some cells contained mostly specific granules of
a normal appearance, with an intact crystal core and matrix. In the specimen to which the cells depicted in Figures
2 and 3 belong, 21 out of 62 cells (34%) showed a normal
ultrastructure, whereas the rest (66%) showed clear, widespread alterations of their specific granules.
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In granules with an intact core and matrix, ECP labeling was almost exclusively associated with the matrix. Instead, in granules in which the core had disintegrated, ECP labeling was found scattered over the whole cross section of the granules. Partially dissolved crystal cores also showed labeling for ECP (Figure 3). In addition, ECP was also observed in the vicinity of partially emptied granules (Figures 2a and 2b; darker areas). In cross sections cutting through the center of the cell, where Golgi apparatus and vesicles but not specific granules are present, labeling for ECP was also detected (Figures 2a and 2c). In some cells the presence of gold particles was observed associated with the nuclear envelope and between nucleus and plasma membrane, in an area devoid of granules (Figure 3). No gold particle was observed on nuclei or other leukocytes present in the specimen, indicating that ECP labeling was specific.
Subcellular Fractionation of Eosinophil Organelles
The second approach to examine the intracellular distribution of ECP in eosinophils was subcellular fractionation on sucrose density gradients.
Characteristic distribution profiles of ECP and organelle markers of eosinophils from an allergic subject during the pollen season are shown in Figure 4. Fractions of density from 1.16 to 1.28 g/ml included one major and three minor peaks of hexosaminidase activity. Peaks I and II contained specific granules, as shown by EM (Figure 5a). Peak III contained small and partially empty specific granules, mitochondria, and double membrane vesicles (Figure 5b). The equilibrium density of peak M corresponded to lysosomes and mitochondria, which could in fact be observed by EM (Figure 5c). Fractions of densities from 1.095 to 1.17 g/ml contained most of the activity of alkaline PDE (plasma membrane marker) and a part of the peak of mannosidase activity (Golgi marker) (Figure 4b). The morphology of the structures found in fractions of densities from 1.11 to 1.20 g/ml is shown in Figures 5c-5e. Mitochondria, lysosomes, and different vesicular organelles (single- and double-membrane vesicles and tubule-vesicular structures) could be seen in different areas of the sample. The less-dense fractions of the gradient (from 1.04 to 1.10 g/ml) included peaks of free protein (cytosolic protein), albumin (CD11b-containing vesicles), and part of the mannosidase peak (Golgi) (Figure 4c).
In healthy subjects (Figure 6), the bulk of ECP and EPO were localized in the high-density region associated with peaks I and III+II and to a much smaller extent with peak M. In this separation peaks II and III overlapped. Fractions of lower densities contained small concentrations of ECP and EPO. These subcellular distributions were the same during and out of the pollen season (not shown).
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In allergic subjects, seasonal changes in the distribution of ECP and EPO were observed (Figure 7). During the pollen season, the amounts of ECP and EPO increased in fractions of densities from 1.11 to 1.16 g/ml, creating an additional peak. The distribution of both proteins in February was similar to those of healthy subjects. The profiles shown in Figure 7 correspond to eosinophils from the same subject as those shown in the electron micrographs of Figures 1-3.
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Comparison of ECP and EPO Amounts in Fractions of Low Density
For accurate comparison of the amounts of ECP and EPO in low-density subcellular fractions of cells from different subjects and at different times of the year, we calculated the contents of ECP and EPO in pools of fractions of densities from 1.11 to 1.16 g/ml, which contain membrane-bound structures but are devoid of specific granules. The median contents of ECP and EPO in the low-density organelle pools from healthy individuals were 230 ng/106 cells (n = 9) and 150 ng/106 cells (n = 5), respectively (Figure 8), regardless of the time of year in which the blood samples had been obtained.
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In allergic subjects, the median content of ECP in this pool was 188.4 ng of ECP/106 cells before the pollen season (Figure 8), which increased during allergen exposure to 558 ng/106 cells. The changes were similar for EPO, with an even more pronounced increase in the low-density pool, from 294 ng/106 cells to 1,423.8 ng/106 cells during the season (Figure 8).
Subcellular Fractionation of Eosinophils of Different Densities
The increase of matrix proteins observed in the low-density region of gradients (Figures 7 and 8) corresponds to the eosinophil population as a whole. In allergic patients, eosinophils are known to be heterogeneous regarding their density (9). Because a correlation between the number of hypodense eosinophils and serum levels of eosinophil granule proteins has been reported (10), which suggests that degranulation may be the cause of hypodensity, we undertook the investigation of the distribution of granule matrix proteins in eosinophils of different densities.
Eosinophils obtained from one allergic subject during the pollen season were separated on a Percoll gradient. Cells of five different densities were obtained and examined by light microscopy after conventional staining with H&E, which showed that they presented the normal appearance of mature cells with the exception of a few apoptotic cells in the least-dense bands, Figures 9a and 9b, bands 1 and 2, respectively. The eosinophil distributions in the different bands and their characteristics are in Table 1. Each of these five cell populations was subcellularly fractionated on a sucrose gradient II (see MATERIALS AND METHODS) to achieve a better resolution of the different types of granules. The distribution of ECP is shown in Figure 9. Band 5 cells (Figure 9e) showed an ECP profile with three peaks (I, II, and III) of specific granules and small amounts of ECP in fractions of lower densities. In band 4 cells (Figure 9d), peak I decreased dramatically while peak II remained unchanged and peak III increased, as did the region of fractions of densities 1.07 to 1.21 g/ml which includes plasma membranes, Golgi apparatus, mitochondria (M), and lysosomes, as shown by the marker distributions and the EM characteristics already described. The ECP profiles of cells from bands 1, 2, and 3 (Figures 9a-9c, respectively) did not differ significantly from each other and showed smaller total amounts of ECP per cell and decreases in Peaks III and M as compared with band 4 (Figure 9d). A small amount of ECP was still found in the region of densities 1.12 to 1.17 g/ml colocalizing partially with alkaline diesterase.
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The subcellular distribution of matrix proteins other than ECP (EPO and hexosaminidase) in eosinophils of different densities showed both similarities and some remarkable differences, as Figure 10 illustrates. As with ECP, the amounts of EPO and hexosaminidase in peak I of the specific granules of hypodense eosinophils (bands 3 and 4; Figures 10a and 10b, respectively) were greatly decreased as compared with normodense cells (band 5; Figure 10c). A prominent peak of hexosaminidase, but neither of ECP nor of EPO, was apparent in the very low density region (1.05 to 1.13 g/ml) of all three gradients which colocalizes with albumin (vesicle marker). The content of hexosaminidase in fractions of density from 1.17 to 1.21 g/ml was substantially higher in normodense (band 5; Figure 10c) than in hypodense (bands 3 and 4; Figures 10a and 10b, respectively) eosinophils. ECP was increased in the same density range but only in band 4 cells. On the contrary, the content of EPO in that region of the gradient was always low for all cell densities. EPO increased in fractions of density from 1.12 to 1.17 g/ml in bands 3 and 4.
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The distribution profiles of bands 1 and 2 cells (not shown) were very similar to that of band 3 (Figure 10a).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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This study demonstrates for the first time that eosinophils from the peripheral blood of subjects with active allergic disease show signs of PM. We show that hypodense eosinophils of an allergic subject have less ECP and other matrix proteins in the specific granules and more in compartments outside the granules.
The morphology of eosinophils from nonsymptomatic
allergic subjects did not differ from that of healthy individuals (20). However, during the pollen season the eosinophils from allergic subjects showed dramatic changes. The
presence of specific granules showing losses of crystal
cores and matrices to different extents is similar to the picture defined by Dvorak and coworkers (1) as PM. We considered carefully the possibility of artifacts having been
caused by the fixation, embedding, or sectioning procedures used. We believe that the differences in the ultrastructure of specific granules actually reflect their activation state because of the reasons that follow. We observed
granule structures at different stages of emptyingfrom
loosening of the matrix and core to more advanced stages
up to complete emptyingoften all within the same cell.
We also observed cells containing affected and unaffected
granules adjacent in the same section. In addition, eosinophils from both healthy and allergic subjects out of the season showed a clear predominance of cells containing
unaffected specific granules although they had been fixed
and embedded in the same way as cells from allergic subjects during the season. In support of our view, cells other
than eosinophils present in the specimens, such as neutrophils, did not show alterations in the structure of their
granules either during or after the season.
The pollen season-related changes in eosinophil morphology and intracellular distribution of ECP may be a consequence of the cascade of proinflammatory events and increased production of T-helper 2-type cytokines that follows allergen exposure (21, 22).
We showed that granule matrix proteins such as ECP are not present exclusively in the specific granules. Normal granules with intact crystal cores and homogenous matrices showed ECP associated with the matrix, as previously described (20). When a core lost its crystalline character, labeling for ECP was found in the partially dissolved areas of the core, which suggests that ECP moved from the matrix. ECP was also present in the vicinity of partially emptied specific granules, in areas of the cytosol with an electron density similar to that of the crystal core of normal granules. The presence of ECP in the regions of dissolving crystal cores is therefore likely to be a consequence of its release from the granule matrix.
Specific granules seemed to degranulate in two different ways. In some the crystal core dissolved and appeared outside the granule while the matrix was still present in the granule, although with a lower electron density. Other granules lost their matrices and showed ragged losses in their cores, whose crystalline character was still preserved. It is not known how this differential emptying of the granules may occur.
The question arises about the transport of ECP as part of the mechanism of PM in circulating eosinophils; specifically whether it is transported within vesicular structures (as shown for EPO by histochemical methods [1]) or is present as a free protein in the cytosol. The paraformaldehyde fixation and Lowicryl embedding technique we used for whole-cell preparations is highly satisfactory for the preservation of antigenicity but does not allow the visualization of small structures such as transporting vesicles, due to a low membrane contrast. Nevertheless, in subcellular fractionation experiments we observed that the ECP and EPO in compartments outside the specific granules did not colocalize with free cytosolic proteins but with the plasma membrane marker alkaline PDE. The electron microscopic examination of this extragranular pool showed the presence of a variety of vesicles that strongly resemble those described by Dvorak and colleagues (1) as containing EPO in whole cells. Inasmuch as no marker indicating the identity of these vesicles is known, we could not show their localization in gradients using biochemical methods.
The ECP and EPO present at low equilibrium densities overlap well with the alkaline PDE marker for plasma membranes. Nevertheless, the electron microscope pictures showed very weak, if any, ECP immunolabelling of plasma membranes. This could be due to ECP and EPO being associated with structures of buoyant density similar to that of plasma membranes, or alternatively to the fact that membranes of vesicles transporting ECP and EPO contain PDE. The presence of alkaline PDE in the specific granules suggests the possibility of membrane transport between the plasma membrane compartment and the specific granules. Altogether, it seems more likely that ECP and EPO leave the specific granules within membrane-bound structures rather than as free proteins. The functional characteristics and role of these structures remain to be determined.
Whereas EM gives an insight into events at the level of single cells, the technique of subcellular fractionation allows us to quantify the average amounts of ECP and EPO present in different cell compartments of heterogeneous cell populations, with the limitation of the equilibrium density superimposition of different organelles. By subcellular fractionation, we observed an increase in the amounts of ECP and EPO in the vesicular compartment outside the specific granules during the pollen season. This can be interpreted as an increase in the transport of these proteins from the specific granules toward the plasma membrane.
EM images suggested that eosinophils were morphologically altered to different degrees. Some cell cross sections showed most of the specific granules with a normal appearance whereas others showed granule alterations to different extents. The intensity of ECP labeling also seemed to vary between cells. These findings suggest the existence of subpopulations of eosinophils degranulated to different degrees.
It has been shown previously that the number of hypodense eosinophils in peripheral blood correlates with the activity of allergic disease (9). We hypothesize that a progressive degranulation of eosinophils during allergen exposure may give rise to hypodense eosinophils. Indeed, a reduced content of eosinophil granule proteins in the specific granules was observed in eosinophils of density lower than 1.092 g/ml (bands 1-4) compared with normodense cells (band 5). The increase of ECP and EPO in the low-density subcellular fractions from band 4 cells compared with either band 5 or band 3, 2, and 1 cells could be an indication of ongoing degranulation. The lower content of ECP and EPO in the low-density gradient region of band 3, 2, and 1 cells could mean that these cells have already degranulated, whereas the similarly lower content in the same gradient region of band 5 cells could indicate that they have not yet degranulated.
The differences observed in the subcellular distribution of ECP and EPO between hypo- and normodense eosinophils could have also been the result of the low-density cells being immature. We excluded this possibility, though, on the basis of the light microscopy observations, which showed that hypodense cells had the morphologic features of mature cells.
Neither the ECP distribution nor its total amount in cells of densities < 1.086 g/ml (bands 1, 2, and 3) differed significantly from each other. A lower eosinophil density may be due to other factors such as an increased number of intracellular lipid bodies or changes in the relative proportions of cell to granule surface due to granule swelling, as reported previously (23).
The three proteins measured (ECP, EPO, and hexosaminidase) had different subcellular distributions compared with each other in the compartment outside the specific granules, according to cell density. These differential distributions suggest the existence of different mechanisms for the intracellular transport of individual proteins.
The ECP distribution in cells of density between 1.092 to 1.100 g/ml was similar to that observed in the mixture of eosinophils of all densities from normal subjects, whereas the distribution in cells of density lower than 1.092 g/ml was like that of allergic subjects. This suggests that the changes in ECP profiles in eosinophils of mixed densities during the pollen season could be due to an increase of hypodense eosinophils in the circulation.
Eosinophil degranulation could theoretically occur in
the blood, in the bone marrow, or both. It has been shown
that eosinophil progenitors can degranulate during the maturation process, as suggested by experiments using cells in
culture (4, 24). Another possibility is the gradual release of
proteins from eosinophils during their lifetime in blood.
Because many eosinophil granule proteins have strong cytotoxic properties, the matter of their elimination from the
circulation is raised. In the case of ECP, it has a half-life of
65 min in circulation (18), and its potentially harmful cytotoxic effects are counteracted by its forming complexes with
2-macroglobulin (25) and negatively charged molecules such as heparin (26).
ECP is synthesized mainly during eosinophil maturation in the bone marrow (27, 28). However, it has been shown that peripheral blood cells can also synthesize ECP, as shown at messenger RNA levels in hypereosinophilic patients (29) and healthy subjects (J. Byström and associates, unpublished findings). By EM we observed the presence of ECP associated with the envelopes surrounding cell nuclei, which is the place of protein synthesis, and to the Golgi apparatus in the central parts of cells. By subcellular fractionation, we detected ECP partially colocalizing with mannosidase, a marker for the Golgi system. This suggests that peripheral blood eosinophils in allergic subjects may retain the ability to synthesize ECP.
We conclude that natural allergen challenge of sensitive subjects causes PM of peripheral blood eosinophils, as shown by their morphology and by the presence of ECP outside specific granules both by EM and subcellular fractionation. We have presented evidence that the heterogeneity of eosinophils regarding their density is related to their degree of degranulation. Our findings suggest that PM may be the mechanism of mobilization of eosinophil granule proteins in the peripheral circulation that could explain the differential levels of plasma and serum eosinophil granule proteins observed in a variety of conditions.
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Footnotes |
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Address correspondence to: Malgorzata Karawajczyk, Dept. of Medical Sciences, Clinical Chemistry, University Hospital, SE-751 85 Uppsala, Sweden. E-mail: Malgorzata.Karawajczyk{at}klinkem.uu.se
(Received in original form November 17, 1999 and in revised form June 1, 2000).
Acknowledgments: This project was supported by the Medical Research Council, Vårdal Stiftelsen, Uppsala University, and Bror Hjerpsted Stiftelse. The authors thank Mr. Anders Ahlander for his excellent technical assistance, Ms. Ulrike Spetz-Nyström for help in collecting patients' samples, and Dr. Xiao Xu for help with purifying eosinophils.
Abbreviations ECP, eosinophil cationic protein; EM, electron microscopy; EPO, eosinophil peroxidase; EPX, eosinophil protein X; PBS, phosphate-buffered saline; PDE, phosphodiesterase; PM, piecemeal degranulation; RT, room temperature.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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1. Dvorak, A. M., S. J. Ackerman, T. Furitsu, P. Estrella, L. Letourmeau, and T. Ishizaka. 1992. Mature eosinophils stimulated to develop in human-cord blood mononuclear cell cultures supplemented with recombinant human interleukin-5. II. Vesicular transport of specific granule matrix peroxidase, a mechanism for effecting piecemeal degranulation. Am. J. Pathol. 140: 795-807 [Abstract].
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