INTRODUCTION

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Keywords: eosinophils; steroids; apoptosis; transmission electron microscopy

Accumulation of eosinophils in peripheral blood and tissues is a major feature of inflammatory airway diseases such as asthma and rhinitis (1). Much is known about bone marrow production of eosinophils, their release into the blood, and subsequent migration into the lung tissue (4). Recently, attention has also been drawn to the mechanisms of resolution of airway eosinophilia. The focus has exclusively been on eosinophil apoptosis, a mode of death by which cells may disappear without inducing any further inflammatory reaction (5). Purified eosinophils that are examined in vitro die spontaneously through apoptosis. Steroids increase the occurrence of eosinophil apoptosis in vitro (6, 7) and when growth factors have been depleted from the cell medium eosinophil apoptosis is intensified (8). Based on the in vitro data, it is widely held that eosinophil apoptosis followed by macrophage engulfment is important for the resolution of airway inflammation (11) and that induction of apoptosis of airway tissue eosinophils is a major facet of the pharmacology of antiasthma steroids (12, 13).

Because cell phenotypes and the molecular milieu may differ greatly between in vitro and in vivo (14), new hypotheses that are based on in vitro data need to be tested in vivo, critically and unassumingly. With regard to eosinophil apoptosis, its occurrence in diseased airway tissues in vivo, with or without steroid treatment, has not been compellingly demonstrated (14). It is clear that apoptotic eosinophils occur in the airway lumen (15) but the luminal eosinophils may not necessarily mimic those present in the airway tissue (14). Also, migration into the lumen can be viewed as a mode of eliminating eosinophils from diseased airway tissue (14). Indeed, considering its potential capacity as a clearance mechanism, it is surprising that luminal entry has received little attention as an alternative fate of airway tissue eosinophils to apoptosis (14).

A rat Sephadex model was considered well-suited for a study of eosinophil clearance. The lung pathophysiology in this model is characterized by sustained proteinaceous edema and pronounced eosinophilia, both features being manifest within 24 h after tracheal instillation of a single dose of Sephadex beads (16). Previous work involving steroids in this or other models of airway inflammation has focused largely on effects of pretreatment with these drugs. However, efficacy regarding effects on established inflammation can not be judged simply by extrapolating from observations made by using prophylactic drug treatment. In this study, in which drug treatment starts 24 h after Sephadex challenge (when a tissue eosinophilia is already established), we have selected a dose of steroid that, by pretreatment, completely inhibits the Sephadex-induced edema and eosinophilia (16). The extensive proapoptotic action of steroids on eosinophils in vitro is fully established within 24 h (6, 7). Hence, a major focus of the present study is on features of airway tissue eosinophils at time points up to 48 h after instituting the steroid treatment. The 24-h and 48-h time points are further selected for the present analyses of luminal entry of eosinophils and of features of luminal eosinophils, including the possibility that steroids may affect the luminal entry of eosinophils and increase eosinophil apoptosis in the lumen.

Because almost nothing is known about the time course of effects of steroids on established pulmonary eosinophilia, a separate part of this study examined Sephadex-challenged animals for 9 d. The aim of this set of experiments was to find and study a phase when resolution of Sephadex-induced eosinophilia occurred, either spontaneously or as a result of steroid treatment. In studies of apoptotic eosinophils in vivo, morphology is essential (19). Hence, it is vital to employ methods that clearly identify the morphologic changes that are characteristic of apoptosis. In this study, we have employed transmission electron microscopy (TEM) in particular, the gold standard, for identifying apoptotic cells and we have used light microscopic techniques, including terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) staining (22, 23).

Our data suggest that eosinophil apoptosis is not induced by steroid treatment in vivo, neither acutely nor by prolonged treatment when resolution of tissue eosinophilia occurs. Indeed, we find that eosinophil apoptosis is exceedingly rare in the rat pulmonary tissues in vivo, irrespective of steroid treatment. However, luminal entry, in part followed by eosinophil apoptosis in the lumen, emerges as a mode of clearing airway tissue eosinophils. Importantly, the migration of eosinophils into the airway lumen was not reduced by steroid treatment.

	METHODS

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Sephadex and Glucocorticosteroid Administration into Rat Lungs

Male Sprague-Dawley rats (220 g) (Möllegard Breeding Centre Ltd., Ry, Denmark) were used. The study was approved by the ethics committee of the Faculty of Medicine at the University of Lund. Under anesthesia with enflurane (Abbot, Kista, Sweden) supplied with O₂ and N₂O, intratracheal instillations were made with either saline (n = 3) or Sephadex beads (n = 6) (G-200 superfine, 5 mg/ml, suspended in sterile saline; Pharmacia, Uppsala, Sweden) to establish an eosinophilic and edemogenic inflammation as previously described (16, 24). Sephadex G-200 is composed of cross-linked dextran beads to which rats have an endogenous hypersensitivity (25). When the inflammation was established (24 h later), budesonide (n = 6) (1 mg/kg dissolved in sterile saline; AstraZeneca, Lund, Sweden) or saline was instilled intratracheally once daily for 2 d. A complementary experiment was made with prolonged steroid treatment for 9 d.

Determination of Cells and Tumor Necrosis Factor-alpha (TNF-) in the Bronchoalveolar Lavage Fluid (BALF)

After killing the rats with intraperitoneal injection of sodium pentobarbital 5 ml/kg (Mebumal vet., Nordvacc, Sweden), lungs were removed and bronchoalveolar lavage (BAL) performed (16, 24). Cytospin slides of BALF cells were stained with May-Grünwald-Giemsa and differential cell counts were carried out. BALF TNF- concentrations were determined by rat TNF- ELISA (R&D Systems, Minneapolis, MN).

Tissue Samples

From each animal, three tissue samples were taken from the superior lung lobe at the level just below the root of the lung. For histochemistry, samples were fixed overnight in Stefanini's fixative (2% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer pH 7.2). Samples for paraffin sections were fixed overnight in buffered 4% paraformaldehyde (pH 7.2). Additional samples were placed overnight in a mixture of 3% formaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2 and used for TEM analysis.

Histochemistry

Eosinophils in lung tissues were detected by histochemical visualization of cyanide-resistant eosinophil peroxidase (EPO) (26, 27). Eosinophils were quantified in the peribronchial tissues of lung specimens in a blinded manner and expressed as number of eosinophils/0.1 mm² tissue area.

Detection of Apoptotic Cells in Lung Tissues with the TUNEL Technique

Sections were cut (4 µm), deparaffinized, pretreated with proteinase K (20 µg/ml; Sigma, Stockholm, Sweden) for 15 min at room temperature. Apoptotic cells were visualized using the TUNEL technique (28) (ApopTag fluorescein; Intergen Company, New York, NY). Thymus from steroid-treated rats was used as a positive control. No staining was evident in negative controls when omitting the terminal deoxynucleotidyl transferase (TdT) enzyme. Slides were counterstained with the DNA-binding stain propidium iodide to reveal pyknotic nuclei as well as the total number of cells. Apoptotic eosinophils were defined as both chromotrope 2R-positive (29) and TUNEL-positive cells exhibiting apoptotic morphology (small cells with condensed nuclei).

TEM

The fixed specimens were handled as previously described (27). The sections were examined using a Philips CM-10 transmission electron microscope. To properly assess eosinophil apoptosis in vivo, we used detailed information on apoptotic growth factor-depleted human blood eosinophils. The ultrastructural criteria for eosinophil apoptosis were eosinophils displaying cell shrinkage and nuclear chromatin condensation (30).

Statistical Analysis

All data are presented as mean ± SEM. To determine statistical significance between treatment groups, Wilcoxon-Mann-Whitney test was performed and p < 0.05 was considered significant.

	RESULTS

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Lung Edema

Sephadex administration produced pulmonary edema that was fully established within 24 h as shown by increased lung weights. Budesonide treatment promptly and durably resolved the established inflammatory edema (Figure 1). Body and thymus weights were reduced (p < 0.001, data not shown), indicating and confirming (16, 24) significant systemic effect of the present steroid treatment.

View larger version (19K): [in this window] [in a new window]   Figure 1.   Provocation with Sephadex beads produced sustained lung edema (increase in lung weight). Budesonide treatment promptly resolved the Sephadex-induced inflammatory edema. Data are mean ± SEM. ***p < 0.001, **p < 0.01 indicates difference between Sephadex and saline treatments. (***) or (**) indicates difference between budesonide-treated and placebo-treated Sephadex-challenged animals.

Sephadex-induced Leukocyte Infiltration into Lung Tissue

Sephadex administration produced a marked infiltration of leukocytes. Granulomas were formed around large bronchi and vessels, as well as in association with the Sephadex beads. The cellular infiltration was characterized by a prominent and sustained tissue eosinophilia (Figure 2). The short-term budesonide treatment had no effect on the established tissue eosinophilia (Figure 2). In the complementary experiments where the steroid treatment was prolonged, the tissue eosinophilia was reduced at 96 h and 240 h after the Sephadex challenge (i.e., 72 h and 216 h after start of steroid treatment) (Figure 2). Sephadex administration also induced a transient neutrophil infiltration in the lung tissue, which peaked at 24 h. The Sephadex-induced tissue neutrophilia was reduced (p < 0.05) by budesonide.

View larger version (15K): [in this window] [in a new window]   Figure 2.   Sephadex provocation-induced peribronchial tissue eosinophilia was not resolved by short-term (2-d) steroid treatment. An additional experiment (dotted lines) showed that the tissue eosinophilia was resolved by prolonged budesonide treatment. Data are mean ± SEM. **p < 0.01 indicates difference between Sephadex and saline treatments. (***) or (**) indicates difference between budesonide-treated and placebo-treated Sephadex-challenged animals.

BAL

The total number of BALF leukocytes was increased in the Sephadex-treated animals (Figure 3A). Neutrophils peaked at 48 h and then declined (Figure 3A) whereas lymphocytes increased until 48 h and remained high at 72 h (Figure 3A). Total BALF cells and lymphocytes were decreased by 2 d of budesonide treatment (Figure 3A). Two days of treatment with budesonide was not sufficient to reduce the established BALF eosinophilia (Figure 3A). Instead, a tendency toward a higher proportion of BALF eosinophils was seen in the steroid-treated animals (Figure 3B). Neutrophils and eosinophils with morphologic signs of apoptosis were frequently observed in the BALF. The number of apoptotic BALF neutrophils (data not shown) and eosinophils (Table 1) was not changed by the present steroid treatment. TNF- concentrations in BALF reached a peak 30 h after Sephadex administration (0.83 unit ± 0.14 compared with saline-treated animals 0.10 ± 0.01; p < 0.01). The TNF- concentrations correlated with a general increase in apoptotic cells in the lung tissue (r = 0.82, p < 0.001). Budesonide treatment did not influence the BALF concentrations of TNF- (peak level 0.87 unit ± 0.19).

View larger version (22K): [in this window] [in a new window]   View larger version (24K): [in this window] [in a new window]   Figure 3.   (A) Total number of cells and large mononuclear cells (LMNC), neutrophils, eosinophils, and lymphocytes in BALF after treatment with saline, Sephadex, or Sephadex + budesonide (bud). Data are mean ± SEM. **p < 0.01 indicates differences between Sephadex and saline treatments. (**)p < 0.01 indicates difference between budesonide-treated and placebo-treated Sephadex-challenged animals. (B) Percent eosinophils in BALF after Sephadex provocation and budesonide treatment.

Apoptotic Cells in Lung Tissue

Sephadex produced a transient increase in the total number of TUNEL-positive cells in the lung tissue (Figures 4 and 5), and these cells were not promptly removed by macrophage engulfment. Apoptotic eosinophils were not detected. Budesonide reduced the total number of TUNEL-positive cells (Figures 4 and 5). None of the TUNEL-positive cells in the peribronchial and perivascular tissue was identified as an eosinophil in the budesonide-treated animals.

View larger version (13K): [in this window] [in a new window]   Figure 4.   Provocation with Sephadex beads produced a general increase in the total number of apoptotic cells (TUNEL-positive) in lung tissue. Steroid treatment reduced the number of apoptotic cells. Data are mean ± SEM. ***p < 0.001 indicates differences between Sephadex and saline treatments. (***)p < 0.001 indicates difference between budesonide-treated and placebo-treated Sephadex-challenged animals. Left bar in each group of three indicates saline; middle bars indicate Sephadex; right bars in each group indicate Sephadex + budesonide.

View larger version (25K): [in this window] [in a new window]   Figure 5.   Fluorescence micrographs demonstrating TUNEL-positive cells (green fluorescence) in steroid-treated thymus tissue (A), serving as a positive control. Nuclei of nonapoptotic cells are counterstained with propidium iodide (red fluorescence). Saline-treated rat lungs contained few TUNEL-positive cells in the peribronchial tissue around large bronchi (B). The number of TUNEL-positive cells increased after Sephadex provocation (C). Scale bar = 100 µm.

TEM Analysis of Lung Tissue and Airway Lumen Cells

TEM analysis of cross-sectioned airways revealed that in the airway lumen apoptotic eosinophils occurred frequently (Figure 6A and Table 1). However, the tissue eosinophils were viable and intact (Figure 6B). Importantly, only one occasional apoptotic eosinophil was seen in the Sephadex-provoked lung tissue. This archetypal apoptotic eosinophil occurred in a granuloma formation (Figure 6C). Also, only one of 678 TEM-analyzed eosinophils was observed inside a macrophage, and this particular eosinophil exhibited no sign of apoptosis. The steroid treatment did not increase the occurrence of apoptotic eosinophils in the lung tissues (Table 1). Apoptotic cells other than eosinophils, for example, neutrophils and type II pneumocytes as well as a few macrophages containing apoptotic cell material (Figure 7) were observed in the lung tissue. In general, with most of the observed apoptotic cells there were little signs of acute engulfment by the macrophages that abounded in the lumen (Figure 3A) and the tissue. The steroid treatment did not appreciably increase the occurrence of engulfed apoptotic cells.

View larger version (139K): [in this window] [in a new window]   Figure 6.   Electron micrographs of eosinophils in Sephadex-treated lungs. Apoptotic eosinophils with reduced size and condensed nucleus were frequently observed in the airway lumen (A, arrow). Almost all tissue eosinophils were viable, displaying dark specific granules (B). An occasional apoptotic eosinophil was detected in the tissue (C ). Chromatin condensation in the apoptotic eosinophils generally led to a distinct separation between euchromatin and heterochromatin (see the condensed nuclei in A and C ). Original magnification: A and B = ×6,600 and C = ×15,500.

View larger version (195K): [in this window] [in a new window]   Figure 7.   Electron micrograph of an apoptotic neutrophil being phagocytosed by a lung tissue macrophage.

	DISCUSSION

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Using a Sephadex model of established lung eosinophilic inflammation, we demonstrate that steroid treatment resolves pulmonary edema without inducing eosinophil apoptosis. Indeed, we could not find any evidence of steroid-induced eosinophil apoptosis even after several days of treatment when the established tissue eosinophilia was declining. Apoptotic eosinophils in the lung tissues in vivo, although demonstrated for the first time by TEM in this study, are exceedingly rare also without steroid treatment. Contrasting the absence of significant eosinophil apoptosis in the lung tissue, eosinophils are clearly eliminated by migration into the airway lumen where they do undergo apoptosis. The present data, which elucidate the fate of airway tissue eosinophils and the action of airway steroids in vivo, are at variance with current notions, which are based on in vitro observations, but are in accord with several scattered findings in human diseased airway tissues.

TEM Analysis and Validation of TUNEL Staining of Apoptotic Cells

The present study confirmed that a single Sephadex challenge induced pronounced lung eosinophilia and showed that this feature was well sustained for more than a week. Hence, the present model seemed well suited for the study of clearance of established lung tissue eosinophilia. To investigate cell death by apoptosis, we chose three independent techniques: light microscopy with May-Grünwald-Giemsa staining, TUNEL staining, and TEM. At the light microscopic level, apoptotic cells may be recognized by their typical pyknotic nucleus and reduced cell volume. These features were also our criteria for identification of apoptotic cells in cytospin slides from BALF. TEM ought to have a higher sensitivity than light microscopy because early apoptotic changes can be identified at the ultrastructural level. We thus demonstrated in the present study that TEM reveals about twice as many apoptotic eosinophils (in the airway lumen; Table 1) as detected by light microscopy. These data support the view that TEM is the gold standard for identification of apoptotic cells (14, 22).

Several molecular approaches have been devised for detection of apoptotic eosinophils in tissues. The TUNEL technique, which is based on enzymatic incorporation of labeled nucleotides at sites where DNA fragmentation has occurred, is commonly used (28). However, the TUNEL staining is not specific to apoptotic cells (e.g., necrotic cells also exhibit DNA fragmentation). Indeed, unless combined with convincing demonstrations of apoptotic morphology, the TUNEL technique is not valid (22, 23). Also, depending on the concentration and incubation time of the enzymes and nucleotides used, TUNEL may fail to stain even truly apoptotic cells or, more commonly, may stain virtually all cells in the tissue irrespective of apoptosis (14). Hence, in this study our TEM analysis served as complement to, as well as validation of, the TUNEL technique. We further used steroid-treated thymus tissue as a positive control, and could demonstrate appropriate TUNEL staining in a tissue where a large proportion of the thymocytes is apoptotic and hence, should be TUNEL-positive (31).

Test of the In vitro-based Hypothesis That Steroids Cause Acute Apoptosis of Lung Tissue Eosinophils In vivo

Importantly, the present study demonstrated actual, but exceedingly rare, occurrence of eosinophil apoptosis in pulmonary tissues. As discussed in some detail elsewhere (14), several previous reports (32) claiming to have demonstrated apoptotic eosinophils in airway tissues may have contained false-positive observations. Indeed, recent uses of TEM analysis in search of proper apoptotic eosinophils in eosinophil-rich airway tissues (14, 27, 36), including human diseased tissues that had been subjected to steroid treatment, did not reveal any apoptotic eosinophils. Hence, to our knowledge this study provides the first demonstration of an eosinophil with clear apoptotic morphology in respiratory tract tissues in vivo.

The evidence of eosinophil apoptosis in the present model would support its suitability for examination of any drug- induced increased eosinophil apoptosis in vivo. Yet, neither TEM nor TUNEL methods found any increased eosinophil apoptosis during the 48 h of highly effective (antiedema and thymus involution) steroid therapy. Furthermore, this duration of steroid treatment includes and, by far, outlasts the period of time within which massive eosinophil apoptosis is induced in vitro. The lack of steroid-induced eosinophil apoptosis in this part of the present study is thus strikingly at variance with previous in vitro observations.

Because the proapoptotic in vitro action of steroids may be overcome by raising the concentrations of prosurvival cytokines (37), the present observations may, speculatively, be explained by an exaggerated presence of eosinophil survival factors in the Sephadex-provoked lungs. It also seems possible, particularly in view of the almost complete absence of apoptotic eosinophils, that the phenotype of purified eosinophils, that may exist only in vitro, is much more susceptible to apoptotic signals and to the proapoptotic action of steroids than the eosinophils that dwell in airway tissues in vivo (14).

Steroids Produced No Immediate Reduction of Established Tissue Eosinophilia; Similarity to Human Asthma?

The expected consequence of a prompt and widespread steroid-induced eosinophil apoptosis, as previously shown in vitro, would be a clear reduction in tissue eosinophilia. No such reduction was observed in this study. The time course of drug-induced resolution of established airway eosinophilia appears to be a poorly explored field of in vivo research. However, the present observation that steroid treatment rather slowly reduces established airway tissue eosinophilia is compatible with observations in asthma and rhinitis (38). It is a separate matter that pretreatment with steroids will promptly prevent challenge-induced eosinophilia in human allergic airway disease (41). Interestingly, also in the present Sephadex model, pretreatment with budesonide has been reported to inhibit the development of lung eosinophilia (16). This latter efficacy of steroid treatment may be explained by the ability of steroids to inhibit eosinophil recruitment events, including effects on interleukin-5 (IL-5), granulocyte macrophage colony-stimulating factor (GM-CSF), eotaxin, or other steroid-sensitive chemoattractant factors (1, 4, 42, 43).

Search for Apoptotic Tissue Eosinophils at Prolonged Steroid Treatment

To further test the possibility that steroids may induce apoptosis in vivo, irrespective of the time periods devised by the previous in vitro studies (8), an additional series of experiments involving prolonged steroid treatment were carried out. Already 72 h after institution of steroid treatment (96 h after challenge), a decline in tissue eosinophilia was well under way in the treated animals. This would then seem the ideal point of time for finding apoptotic eosinophils in the lung tissue, had apoptosis played a role in the reduction of eosinophil numbers. However, apoptotic eosinophils could not be detected, nor could such cells be detected at a later time point when resolution of the tissue eosinophilia was almost complete. Similarly, there was a lack of apoptotic eosinophils in the Sephadex control lungs that remained highly eosinophilic for the entire duration of the present experiments.

The Absence of Apoptotic Tissue Eosinophils May Not Be Explained by Prompt Removal of Apoptotic Cells through Phagocytosis

Rapid phagocytosis of apoptotic eosinophils has been suggested, again based on in vitro data, to explain why such cells may not be commonly found in tissues (6). However, the present data cannot support that notion. The rare apoptotic tissue eosinophil that was observed in the present study occurred in a lung granuloma (not exposed to steroids). The granuloma formations contained many macrophages that only occasionally exhibited phagocytosis of granulocytes (Figure 7). Because the present histologic examination did not reveal eosinophilic material inside tissue macrophages, it appears unlikely that we failed to see apoptotic eosinophils in this study owing to rapid phagocytosis of such cells. Our observation that noneosinophilic, apoptotic cells, such as the present apoptotic neutrophils, clearly occurred in the lung tissue, and had not been phagocytosed, supports the possibility that we should have detected apoptotic eosinophils had such cells been generated in significant numbers. In addition, we did detect many apoptotic eosinophils in the airway lumen (discussed subsequently) where macrophages also abound. We do not argue that eosinophil apoptosis cannot be induced in vivo. Current experiments involving intra-airway administration of Anti-Fas monoclonal antibody (mAb) in allergic mice have thus demonstrated numerous apoptotic tissue eosinophils (44). Moreover, apoptotic eosinophils have previously been observed in parasite-infected and steroid-treated rat gut (45). Interestingly, Kawabori and coworkers illustrated by TEM the in vivo occurrence of apoptotic eosinophils engulfed and not engulfed by macrophages. Hence, it appears likely that had the same events occurred in the present rat lungs we should have detected them by our TEM analysis. However, as suggested by data obtained in this study, steroid treatment may not induce eosinophil apoptosis in vivo in airway tissues. This finding, and the potential role of luminal entry as an elimination mechanism, has further been confirmed in a mouse model involving eosinophilic airway inflammation evoked by a protein antigen (46).

Sephadex-induced General Increase in Lung Cell Apoptosis, and Its Inhibition by Steroids

Interestingly, the present Sephadex provocation increased the total number of TUNEL-positive cells in the lung tissue. Although many apoptotic cells were devoid of typical morphology for proper cell identification by TEM, apoptotic neutrophils clearly occurred. The neutrophils may, in fact, represent a significant component of the present apoptotic cells in lung tissues: They had a particularly rapid turnover in this study, and steroid treatment, which reduced the total number of apoptotic cells, is known to reduce neutrophil apoptosis in vitro (6). BALF TNF- concentrations that were transiently increased in this study correlated with the number of TUNEL-positive cells. However, further data are needed to confirm an involvement of TNF- in the present Sephadex-induced apoptosis.

Speculatively, other cells with a known high turnover in airway inflammation, including epithelial cells, might also exhibit apoptosis in the present model. It is conceivable that the present Sephadex-induced apoptotic responses reflect increased cell turnover caused by target organ inflammation (19). In a current TEM study, we also observed that apoptotic cells and cell debris occur extensively in the affected mucosa of chronic conditions such as inflammatory bowel diseases (Erjefält et al.; unpublished observations). In accordance with this notion, the present anti-inflammatory action of budesonide was associated with reduced numbers of TUNEL-positive cells. It is inferred that airway challenge-induced apoptosis of noneosinophil cells involves steroid-sensitive processes and that this increased apoptosis potentially reflects the severity of inflammatory processes in the respiratory tract.

Migration into the Airway Lumen "Luminal Entry" as an Elimination Path for Airway Tissue Eosinophils

If eosinophils do not use the apoptotic mechanism, how are lung tissue eosinophils cleared? As demonstrated in this study, and, actually, as supported by a wealth of clinical and experimental data (14), eosinophils dwelling in inflamed airway tissues may simply be eliminated by migration into the airway lumen followed by subsequent final clearance mechanisms. The present analysis of BALF after Sephadex provocation suggests that clearance of eosinophils into the lumen is a continuous process. The luminal entry of eosinophils, as well as of other leukocytes, would be a natural part of the activity of an airway inflammation that involves a continuous flow of leukocytes through the tissue. Luminal entry of leukocytes might especially reflect resolution of active processes, but this latter aspect has not been examined in this study.

Once in the lumen, cells will be mixed with other components of airway discharges and be finally removed by normal physiologic clearance mechanisms such as mucociliary transport or coughing. These events would give little opportunity for cells to travel back into the tissue in large numbers. Furthermore, as demonstrated in this study, a substantial portion of the luminal eosinophils may die through apoptosis. Detailed analyses using TEM revealed that approximately 20% of the luminal eosinophils were apoptotic. This study thus provides the first quantitative data demonstrating that luminal eosinophils undergo apoptosis much more frequently than eosinophils resident in the lung tissue compartment. In accordance, although no apoptotic eosinophils were detected in previous studies of human airway tissues in asthma (15), allergic rhinitis (27, 47), and nasal polyposis (48), apoptotic eosinophils clearly occurred in the lumen in these eosinophilic airway diseases. It would seem an ideal mode of clearance of airway tissue eosinophils: first, tissue escape through luminal entry, and then final removal by a combination of apoptosis and mucociliary transport (Figure 8). The present steroid treatment did not cause any acute (24- to 48-h) reduction in the percentage of eosinophils in BALF, suggesting that essential mechanisms involved in the luminal entry of eosinophils are not inhibited by steroid treatment. Hence, an unimpeded clearance of eosinophils across the epithelial lining, in combination with inhibition of eosinophil recruitment (1, 42, 49), including inhibitory effects of budesonide on bone marrow production and release of eosinophils (4) might be an important facet of the clinical pharmacodynamics of airway steroids (Figure 8). Further studies testing this hypothesis are warranted.

View larger version (44K): [in this window] [in a new window]   Figure 8.   The present data suggest that airway tissue eosinophils in vivo are eliminated by entry into the airway lumen followed by a combination of apoptosis and mucociliary transport (eosinophil apoptosis occurs almost exclusively in the airway lumen). Steroids may not induce eosinophil apoptosis in the tissue, but they permit the clearance into the airway lumen. These features of the in vivo pharmacology of airway steroids, combined with known inhibitory effects on eosinophil recruitment, may explain the present observations on steroid-induced slow resolution of established lung tissue eosinophilia. Interestingly, this scheme of events is compatible with several observations made in human airways in vivo.

In conclusion, this study has demonstrated that exceedingly few tissue eosinophils in inflamed rat lungs are apoptotic. Furthermore, steroid-induced eosinophil apoptosis was not detected. This latter observation is unexpected because the presence of steroids in culture test systems brings about eosinophil apoptosis very effectively within hours. We submit that eosinophils dwelling in the blood-perfused living tissue may be phenotypically distinct from eosinophils in vitro. Importantly, our data indicate an alternative route of elimination to apoptosis because many tissue eosinophils moved into the airway lumen. We further showed that the luminal entry process was well maintained during steroid treatment. As also demonstrated in this study, once in the lumen many eosinophils undergo apoptosis. We thus suggest that luminal entry of eosinophils, followed by apoptosis, may be a major mode of removal of these cells from lung tissues. The reduced tissue eosinophilia observed after several days of steroid treatment may well be explained by inhibition of eosinophil recruitment in combination with an unimpeded clearance of tissue eosinophils into the airway lumen.