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Cytotoxic eosinophil granule proteins are considered important in the pathogenesis of allergic airway diseases such as rhinitis and asthma. To explore the cellular mechanisms behind eosinophil granule release in human allergic airways, 16 symptom-free patients with seasonal allergic rhinitis were challenged daily with allergen during 1 wk. Nasal lavage samples and biopsies, obtained before and 24 h after the last allergen exposure, were processed for immunohistochemical and electron microscopic analysis. The allergen challenges produced nasal symptoms, marked tissue eosinophilia, and an increase in lavage fluid levels of eosinophil cationic protein (ECP). The nasal mucosa areas with intense extracellular immunoreactivity for ECP were associated with abundant free eosinophil granules. Electron microscopy confirmed the free granules and revealed that all mucosal eosinophils were involved in granule release, either by cytolysis (33%) or piecemeal degranulation (PMD) (67%). Resting or apoptotic eosinophils were not observed. Cytolytic eosinophils had less signs of intracellular granule release (p < 0.001) and a higher content of intact granules (p < 0.001) compared with viable eosinophils in the same tissue. This study demonstrates eosinophil cytolysis (ECL) as a distinct mechanism for granule mediator release in human allergic airway mucosa. The nature and extent of the ECL and its product (i.e., protein-laden extracellular granules) indicate that allergen-induced cytolysis is a primary and major mechanism for the release of eosinophil proteins in human allergic airway inflammation in vivo.
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Eosinophil granulocytes are regarded as key effector cells in the pathogenesis of allergic airway conditions such as allergic rhinitis and asthma (1). The specific granules of eosinophils contain potent cytotoxic proteins. Release of these proteins into the extracellular space is believed to cause a variety of tissue disturbances such as epithelial damage (4), extravasation of plasma (5), and airway hyperresponsiveness (6). However, as yet little is known regarding the mechanisms for the release of granule proteins in vivo.
As suggested by variable degrees of electron lucency of granules in eosinophils examined by electron microscopy, there is a gradual release of proteins from the specific granules of intact eosinophils. This phenomenon has been termed piecemeal degranulation (PMD) (9, 10) and has been described in various eosinophilic conditions, including allergic rhinitis (11) and asthma (12). However, knowledge about the frequency of its occurrence and the quantitative morphology of PMD in diseased airways remains incomplete.
Another event that may cause significant release of eosinophil granule products in allergic airway conditions is eosinophil cytolysis (ECL) (15). During this process the cell membrane is ruptured and specific eosinophil granules are liberated into the surrounding tissue (15). Recent work in guinea-pig models has demonstrated that ECL can promptly be induced in vivo and that the release of free granules correlates with the degree of epithelial damage after allergen challenge (16, 17).
Cytolytic ("necrotic") eosinophils and free granules have been depicted in studies examining various eosinophilic conditions (15). In many of these previous studies, either the phenomenon of ECL was not discussed, or the death of tissue eosinophils was regarded as a secondary event caused "accidentally" by spilling of toxic granule products into the cytoplasm of eosinophils undergoing intense PMD (11, 18). However, if cytolysis is actively induced in mucosal eosinophils this might be a major cellular event in the pathogenesis of allergic airway disease and should be evident after allergen challenge in vivo.
We recently observed that the nasal mucosa of patients with seasonal allergic rhinitis exhibited a 30-fold increase in the number of clusters of free eosinophil granules during the active pollen season (21). The present study aims to examine in detail modes of eosinophil granule release, including the occurrence of ECL in human airways after allergen-induced inflammation. The morphology of ECL and free granules has thus been examined and compared with other fates (e.g., apoptosis) and activation (PMD) of eosinophils. To accomplish this we have examined the ultrastructure of mucosal eosinophils in patients with allergic rhinitis before and 24 h after a session of repeated daily experimental allergen exposures during 1 wk (22). We have determined luminal eosinophil cationic protein (ECP) as a complementary index to eosinophil activation. In addition, we have used immunohistochemical techniques to determine the extent of tissue deposition of extracellular ECP and to assess whether engulfment of eosinophils and free granules by CD68+ macrophages is a common feature in allergen-exposed human airways.
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INTRODUCTION
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Patients
Sixteen atopic patients (age 23 ± 2.9, range 15 to 57) with seasonal allergic rhinitis were examined. Inclusion criteria included a history of strictly seasonal allergic rhinitis and a positive skin prick test to grass allergens. Exclusion criteria were a history of chronic nasal disease other than seasonal allergic rhinitis, positive skin prick tests to perennial allergens, drug treatment, and smoking. The study was performed out of the natural allergen season when all the patients were symptom-free, as confirmed at the hospital before the first allergen exposure. The local ethics committee approved the study, and informed consent was obtained.
Study Design and Sampling
The study design is presented in Figure 1. Using a nasal spray device (23), symptom-free patients (symptom scores = 0) were challenged daily with the relevant allergen (purified grass allergen extracts; ALK, Copenhagen, Denmark). The allergen dose (administered as 100 µl spray) was administered at the hospital under supervision. The allergen doses were selected to induce moderate nasal symptoms and are presented in Table 1. The doses were determined in a separate pilot study (unpublished results). From each patient one nasal lavage sample and two mucosal biopsies were obtained before (right nasal cavity) and 24 h after the final provocation (left nasal cavity). Symptoms were scored each study day.
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Nasal lavage was performed by a nasal pool device (24). Briefly, 10 ml 0.9% NaCl were administered into the nasal cavity and were kept there during 10 min before the solution was recollected for analysis of ECP. Nasal biopsies were obtained during topical anesthesia (20 mg tetracain and 0.1 epinephrine) using a cutting punch biopsy forceps (25) with a width of 2 mm. The biopsies were taken from the inferior nasal turbinate about 0.5 cm from its anterior margin. At each occasion two biopsies were obtained from each patient. Biopsies used for immunohistochemistry were immediately placed in phosphate-buffered saline (PBS) containing 4% formaldehyde and 1% picric acid and left for 3 to 4 h at room temperature. Next, the biopsies were rinsed in sucrose buffer and snap-frozen in isopentane. Biopsies for electron microscopy were immediately placed in fixative (1% glutaraldehyde and 3% formaldehyde in PBS) and left overnight at 4° C.
Symptoms
Symptom scores were recorded before and 0 to 24 h (using symptom diary cards) after each allergen exposure as described elsewhere (21). Briefly, the patients were instructed to record their symptoms until the next challenge the following day. The recorded symptoms, i.e., rhinorrhea, nasal blockage, and sneezing were scored on a four-point scale: score 0: no, 1: mild, 2: moderate, 3: severe symptoms. For each day the mean symptoms were calculated for each patient.
ECP Measurements
The fresh nasal lavage samples were centrifuged (105 × g, 10 min, 4° C)
and supernatants were placed in coded vials and frozen at 
30° C
until analysis. ECP concentrations were measured by a commercial
ELISA kit (Pharmacia Diagnostics, Uppsala, Sweden) according to
the instructions provided by the supplier. The coefficients of variations were < 6%.
Immunohistochemistry
Cryosections 5 µm thick were rinsed in Tris-buffered saline (TBS, pH 7.6) and nonspecific binding sites were blocked with normal rabbit serum (dilution 1:10 in TBS; Dako) for 10 min. Sections used for staining of CD68 (a glycoprotein that is present in the plasma membrane and endosomes of activated macrophages) were placed in 1 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0) and heated in a microwave oven at 700 W for 10 s. Sections were incubated with primary monoclonal antibodies overnight at 4° C. Macrophages were visualized by morphological criteria and staining using a mouse anti-human CD68 (dilution 1:50; Dako, Denmark). The monoclonal antibody EG2 (dilution 1:80; Pharmacia Diagnostics, Sweden), which in formalin-fixed tissues recognizes both "resting" and secreted forms of ECP (26), was used to detect intra- and extracellular ECP. After incubation with the primary antibodies, specimens were rinsed in TBS and incubated with a secondary antibody (rabbit anti-mouse, Dako Z0109, dilution 1:25 in 20% normal human serum [NHS]) for 30 min. After washing in TBS, sections were incubated in mouse alkaline phosphatase-antialkaline phosphatase (APAAP) (Dako D0651, dilution 1:50 in 20% NHS) for 30 min, rinsed, and developed using New Fuchsin (Dako) as substrate. Endogenous alkaline phosphatase activity was inhibited by levamisole. The sections stained for CD68 were also subjected to histochemical staining of eosinophil peroxidase (EPO). All sections were counterstained with Harris's hematoxylin, coated with Aqua Perm mountant (484975 Life Sci. International), dried overnight, and mounted in dextropropoxyphene (DPX).
EPO Staining
The distribution of EPO in cryosections was assessed by histochemical staining of cyanide-resistant EPO (16). The sections were exposed to 100 µl incubation solution (PBS pH 7.6: containing 3.3-diaminobenzidine tetrahydrochloride [75 mg/100 ml; Sigma], H2O2 [100 µl/ 100 ml], and NaCN [50 mg/100 ml]) for 3 min at room temperature. After rinsing in tap water the samples were counterstained with Harris hematoxylin and mounted in DPX.
Transmission Electron Microscopy (TEM)
After fixation, biopsies were rinsed in buffer, postfixed in 1% osmium tetroxide for 1 h, and dehydrated in graded acetone solutions and embedded in Polarbed 812 (Polaron, UK). Plastic sections 1 µm thick were cut on an ultratome (Ultracut E; Leica, Germany), stained with toluidine blue and examined in a light microscope (Axioscop; Zeiss, Germany). Based on the light microscopic evaluation, biopsies with a well-preserved morphology and sufficient number of eosinophils were selected for further detailed electron microscopical analysis. A total of six biopsies (all from allergen-exposed patients) were selected. The selection criteria were a marked tissue eosinophilia (defined as more than 10 eosinophils/mm2 subepithelial tissue) and a preserved structure of the mucosa (no morphological signs of crush artefacts). From each biopsy, areas with intact epithelial lining were selected for further electron microscopical analysis. Ultrathin sections (90 nm) were cut and placed on 200-mesh, thin-bar copper grid and stained with uranyl acetate and lead citrate (13). The specimens were examined by a Hitachi transmission electron microscope (H-7000; Hitachi, Japan).
Quantification
EPO-stained eosinophils in cryosections. Subepithelial eosinophil cells were counted to a depth of 0-250 µm from the basal lining of the epithelial basement membrane and expressed as total numbers per 0.1 mm2 tissue. Intraepithelial eosinophils were counted and expressed as cells/0.1 mm2 epithelial area. The epithelial area was calculated by computer-assisted image analysis using NIH Image 1.33 (National Institutes of Health, Bethesda, MD; Macintosh Computer, Cupertino, CA).
Ultrastructural morphology of eosinophils. Each eosinophil was photographed at ×5,000 magnification and later evaluated. Based on morphological criteria the eosinophils were divided into subgroups defined as follows: Resting eosinophils: No ultrastructural signs of activation, i.e., all specific granules had a solid core surrounded by an intact matrix. PMD: Intact cells displaying characteristic changes of specific granules residing within the cytoplasm (10), i.e., occurrence of partly empty intracellular granules with no signs of granule extrusion. Eosinophil exocytosis: Morphological signs of extrusion of "whole," membrane-free specific granules whose membranes have fused with the cell membrane. ECL: Presence of chromatolysis, loss of plasma membrane integrity, and partly dissolved cytoplasm (15). Apoptotic eosinophils: Presence of electron-dense (i.e., black), condensed chromatin, preserved plasma membrane, and nondilated organelles (27, 28). Clusters of free eosinophil granules: Occurrence of three or more identifiable extracellular membranous eosinophil-specific granules grouped together (13, 15). A useful additional criterion was the presence of collagen or elastic fibers amid eosinophil granules.
Quantification of PMD. In all eosinophils each specific granule was evaluated and classified as either an intact granule (with no signs of degranulation, i.e., intact core and matrix) or "activated" (various structural changes due to PMD, e.g., ragged loss of core material, coarsening of the granular matrix, or more or less empty granules) (9). The extent of PMD for each eosinophil was defined as the percent "activated granules" and referred to as the degranulation index for that cell, as described previously (29).
Statistical Analysis
To examine statistical differences between values obtained before and after the allergen exposures, the mean values at each time point were compared. Mean values of ECP concentrations before and after cell numbers and degranulation indices were logarithmically transformed before analysis. Differences among groups were examined by Student's t test or the Mann-Whitney U test. Statistical differences between groups were assumed for p values < 0.05. All statistical tests were performed with Microsoft Excel version 5.0c and Astute version 1.5. All data are presented as means ± SEM.
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RESULTS |
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Symptoms and Nasal Lavage ECP
The allergen exposures produced nasal symptoms in all patients. After the initial allergen exposures, the mean score indicated moderate symptoms which remained at this level throughout the study (Table 1). The allergen exposures resulted in a significant increase in ECP concentrations in nasal lavage samples obtained 24 h after the last provocation (p < 0.05, Figure 2).
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Mucosal Eosinophils
Allergen provocation produced a significant increase in both subepithelial (p < 0.01) and intraepithelial (p < 0.01) numbers of eosinophils (Figures 3, 4a, and 4b). In the biopsies obtained after the repeated allergen challenges eosinophils with morphological alterations resembling cytolysis (e.g., chromatolysis) were scattered and present amid eosinophils with a normal appearance (Figure 4b). Free eosinophil granules abounded in biopsies rich in eosinophils (Figure 4b). Eosinophils with a morphology indicative of apoptosis (e.g., condensed, intensively stained chromatin) were not found.
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Tissue distribution of ECP and EPO. There was an absence of extracellular distribution of EPO and ECP in the preallergen biopsies regardless of the eosinophil content. In contrast, there was an extracellular staining for EPO (Figure 4c) and ECP (Figure 4d) frequently observed in biopsies obtained after allergen challenge. This intense extracellular staining was restricted to tissue areas rich in free eosinophil granules and cytolytic eosinophils.
Phagocytosis of EPO material by macrophages. Double-staining for CD68 and EPO was performed to assess the extent of phagocytosis of eosinophils or free eosinophil granules by macrophages (Figure 4e). No EPO material was observed in CD68-positive cells before allergen challenges. After the allergen exposures, 0.3% of the total EPO-stained eosinophils colocalized with CD68-positive cells (Figure 4f) and this overlap involved 1.5% of the total CD68-positive cells. In the eosinophil-rich biopsies that were analyzed by TEM only one macrophage contained granules with the crystalline ultrastructure characteristic of eosinophil granules.
Ultrastructural Analysis of Mucosal Eosinophils
Eosinophil subtypes. The proportion of eosinophil subtypes in each of the biopsies used for TEM analysis is shown in Table 2. The subtypes were divided on the basis of a cell ultrastructure indicative of one of the following events: PMD, ECL, apoptosis, or resting. Eosinophil cytolysis and presence of clusters of free granules together comprised one-third of the tissue eosinophils. Both the cytolytic eosinophils and the free granules were randomly distributed and frequently seen among intact eosinophils. Many of the extracellular eosinophil granules had retained their granule membranes (Figure 5e). In all patients all the examined eosinophils were in an activated state, either through PMD of intact cells (Figure 5a) or through cytolysis (Figures 5c and 5d, Table 2). Eosinophils with signs of "classic" exocytosis (extrusion of nonmembranous specific granules) or eosinophils displaying characteristic features of apoptosis were absent. However, scattered apoptotic cells other than eosinophils, e.g., neutrophils and B lymphocytes (plasma cells), were observed.
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Degranulation of eosinophils. Several distinct morphological changes were present in the specific granules of eosinophils undergoing PMD (some of which are depicted in Figures 5a and 5b). Budding portions of granules into vesicle-like structures were observed at the borders of the altered granules. In order to express the degree of PMD for each individual eosinophil the percent altered granules was calculated as a degranulation index. No resting eosinophils were observed and detailed analysis of the degranulation indices among intact eosinophils revealed that all eosinophils underwent PMD (i.e., degranulation index > 0, Figure 6). Significant differences in the mean degranulation index were observed between the patients (Figure 6). Eosinophils in individual biopsies generally displayed homogeneity with regard to the degranulation index (Figure 6).
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Degranulation indices in cytolytic eosinophils. To investigate the possibility that ECL occurred independent of an extensive PMD, comparisons of the extent of PMD were made between cytolytic eosinophils and the intact, viable eosinophils residing in the same tissue. Notably, cytolytic eosinophils displayed distinct ultrastructural changes compared with viable eosinophils (Figure 5a). Cytolytic eosinophils had significantly lower mean indices of PMD than neighboring intact eosinophils (Figure 7a). Furthermore, the cell content of intact specific granules was significantly higher in cytolytic eosinophils compared with the intact eosinophils undergoing PMD (Figure 7b).
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DISCUSSION |
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INTRODUCTION
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The present study involving allergen-exposed patients with allergic rhinitis has demonstrated an abundance of cytolytic eosinophils and clusters of free eosinophil granules along with eosinophils exhibiting PMD but which remain intact. The present human in vivo data further indicate that ECL is a primary event that is induced in eosinophils exhibiting few signs of "degranulation" (i.e., PMD) and that ECL might be an exceedingly common event in human allergic airways subjected to repeated allergen exposure. The ultrastructural analyses of the cytolytic eosinophils and their association with intense extracellular immunostraining for ECP provide novel support to the hypothesis that ECL is a major mechanism of granule protein release in the target tissue of human allergic airway conditions (15).
ECL as a Primary Event
Necrosis, as opposed to apoptosis, is generally considered an accidental death in injurious inflammatory processes (28, 30). The present study provides new evidence that cytolysis of airway mucosal eosinophils may not fall into this general category of necrosis. We thus demonstrated that cytolysis was induced in eosinophils that contained particularly high numbers of otherwise intact granules. Hence, the cytolytic eosinophils were not involved in extensive PMD, suggesting that ECL is a primary event independent of, and distinct from PMD. The cytolytic mechanism appears to be particular to eosinophils because cytolytic cells other than eosinophils were never observed. This specificity together with the ultrastructural characteristics of the present ECL excludes the possibility that the ECL merely was a result of an inflammation-induced necrosis of the mucosa or that cytolysis was the random result of mechanical artifacts such as those inflicted during the taking of biopsies. Our data suggest that ECL is actively induced in viable eosinophils during an allergic inflammation. Also in in vitro experiments (in the absence of cytotoxic compounds), purified human blood eosinophils have been shown to undergo cytolysis rapidly, e.g., after stimulation with Sepharose particles coated with surface immunoglobulin A (SIgA) (19) or human plasma (J. S. Erjefält and associates, unpublished results). Furthermore, studies in guinea pigs have demonstrated that ECL is rapidly induced in vivo after allergen challenge (17) or at epithelial shedding-repair processes (16).
Major Fates of Tissue Eosinophils
The present data identify cytolysis as a major fate of human mucosal eosinophils in vivo. Thus, after multiple allergen exposures one-third of the mucosal eosinophils displayed signs of cytolysis. This observation strongly supports the notion that ECL explains our recent observation of numerous clusters of free eosinophil granules in the nasal mucosa in seasonal allergic rhinitis (21). Furthermore, the present data suggest that ECL has contributed to the numerous observations of free membrane-bound eosinophil granules in bronchial tissues of asthmatic subjects (13, 31). ECL may be a general feature of eosinophilic conditions (15) as recently also supported by observations in atopic dermatitis (2) and nasal polyposis (29). The occurrence of ECL in allergic airway diseases may cause the release of large amounts of granule proteins but would also significantly contribute to local clearance of tissue eosinophils. Hence, ECL may explain why high concentrations of ECP may occur in airway diseases when the number of intact tissue eosinophils is low. Also, due to the occurrence of ECL a correlation between eosinophil numbers and allergic airway disease activity, which frequently is sought in clinical studies, should not always be expected.
In contrast to the present abundance of ECL, eosinophils with classic ultrastructural signs of apoptosis were not detected. This result is of significant interest because there is currently a strong focus on the possibility that apoptosis is a major clearance mechanism for eosinophils within human airway tissues (32). In cultured eosinophils cytokines thus prevent apoptosis and this effect is inhibited by steroids (33). Data that directly support the in vivo relevance of this important in vitro research are yet scarce. Apoptotic eosinophils may occur in the airway lumen (32, 34) but this observation merely supports luminal entry of viable eosinophils as a clearing mechanism. The rate of apoptosis and clearance of apoptotic cells in vivo is currently not known. Hence, it may be implied that the paucity of apoptotic eosinophils in human tissues may simply be explained as a rapid phagocytosis and clearance of apoptotic eosinophils by nearby macrophages. However, several findings in this study indicate that our failure to detect apoptotic eosinophils may reflect a true rarity of this shape of death in vivo in allergic airway tissues: First, several cells other than eosinophils were observed to undergo apoptosis. Furthermore, macrophages with engulfed EPO-stained material were exceedingly scarce in areas that otherwise were rich in intact, as well as cytolytic, eosinophils and free eosinophil granules. Our failure to detect apoptotic eosinophils may be explained by the local eosinophil survival factors (e.g., interleukin-5 [IL-5] and granulocyte-macrophage colony-stimulating factor [GM-CSF]) that are likely to be present during the present allergic inflammation (35). A recent ultrastructural study has also failed to detect apoptotic eosinophils in human airway tissues obtained in vivo during effective steroid treatment (36). This observation suggests that further studies examining the frequency and ultrastructure of apoptotic eosinophils in airway tissues during the resolution of an eosinophilic inflammation are highly warranted.
ECL as a Pathogenic Mechanism
Cytolysis of eosinophils in the airway mucosa may have multiple effects on the local tissues. Cytolysis of any cell is by a proinflammatory event (37) caused by the associated leakage of the cell contents in the tissue. Interestingly, the eosinophil granules that were released by the present cytolytic process had largely retained an intact morphology, indicating the possibility that the actual release of the potent granule products takes place late in the cytolytic process, i.e., from extracellular granules. Indeed, in the present study a particularly intense extracellular immunoreactivity for ECP was present in regions rich in free eosinophil granules and the allergen exposures caused elevated luminal levels of ECP. An extensive extracellular immunoreactivity for major basic protein (MBP) among free eosinophil granules has previously been reported in airways from patients who died from asthma (3), and in allergen-challenged, sensitized guinea pigs clusters of free eosinophil granules have been associated with epithelial injury (17). Common to airways exhibiting the in vivo features of ECL is the occurrence of plasma exudation that dynamically distributes nonsieved plasma proteins throughout the mucosa (38). The multiple potent factors in the plasma may thus affect the eosinophil biology in vivo. Interestingly, plasma together with Sephadex particles induces cytolysis of eosinophils in primary culture (Erjefält and associates, unpublished observation).
The present study provides the first quantification of ECL and PMD in human airways subjected to allergic inflammation. The results point out PMD and ECL as major, but distinct, events leading to granule protein release. It is tempting to speculate that ECL represents an activation mode resulting in a relatively immediate and complete granule release. In contrast, PMD provides the possibility of a long-term, and selective (39) release of granular mediators. The latter aspect may agree with the immunoregulatory roles of eosinophils (40) and their granule products (41). Further studies are warranted to establish the relative contribution of PMD and ECL to the release of eosinophil granule products in allergic diseases. Furthermore, the frequency and dynamics of ECL and PMD during various forms of eosinophilic diseases, including the conditions in nonairway tissues where ECL has been depicted (18, 20, 44, 45), have yet to be determined.
Conclusion
In conclusion, the present work contains novel data that identify ECL as a major mode of eosinophil activation during an allergic inflammation. Repeated allergen exposures of patients with allergic rhinitis thus lead to a marked tissue eosinophilia where one-third of the mucosal eosinophils represent cytolytic eosinophils. Two major forms of activation were discerned, PMD and ECL, whereas apoptosis of eosinophils could not be detected, nor was classic exocytosis of eosinophil granules observed. It was further demonstrated that ECL causing extracellular distribution of protein-laden granules is a primary activation mechanism distinct from PMD. It is suggested that ECL is an important mechanism for eosinophil protein release in allergic airway diseases and that ECL may be an important target for future therapeutic strategies.
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Correspondence and requests for reprints should be addressed to Jonas Erjefält, Department of Physiology and Neuroscience, E-Blocket, Lund University Hospital, 221 85, Lund, Sweden. E-mail: jonas.erjefalt{at}mphy.lu.se
(Received in original form September 11, 1998 and in revised form February 8, 1999).
Acknowledgments: Supported by the Medical Faculty, Lund, Sweden, the Vårdal Foundation, the Swedish Medical Research Council, and Astra Draco, Lund, Sweden, and the Lung Pathology Unit, Imperial College School of Medicine (ICSM), London.
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References |
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REFERENCES
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