Anti-Fas mAb-induced apoptosis and cytolysis of airway tissue eosinophils aggravates rather than resolves established inflammation
© Uller et al. 2005
Received: 30 June 2005
Accepted: 08 August 2005
Published: 08 August 2005
Fas receptor-mediated eosinophil apoptosis is currently forwarded as a mechanism resolving asthma-like inflammation. This view is based on observations in vitro and in airway lumen with unknown translatability to airway tissues in vivo. In fact, apoptotic eosinophils have not been detected in human diseased airway tissues whereas cytolytic eosinophils abound and constitute a major mode of degranulation of these cells. Also, Fas receptor stimulation may bypass the apoptotic pathway and directly evoke cytolysis of non-apoptotic cells. We thus hypothesized that effects of anti-Fas mAb in vivo may include both apoptosis and cytolysis of eosinophils and, hence, that established eosinophilic inflammation may not resolve by this treatment.
Weeklong daily allergen challenges of sensitized mice were followed by airway administration of anti-Fas mAb. BAL was performed and airway-pulmonary tissues were examined using light and electron microscopy. Lung tissue analysis for CC-chemokines, apoptosis, mucus production and plasma exudation (fibrinogen) were performed.
Anti-Fas mAb evoked apoptosis of 28% and cytolysis of 4% of eosinophils present in allergen-challenged airway tissues. Furthermore, a majority of the apoptotic eosinophils remained unengulfed and eventually exhibited secondary necrosis. A striking histopathology far beyond the allergic inflammation developed and included degranulated eosinophils, neutrophilia, epithelial derangement, plasma exudation, mucus-plasma plugs, and inducement of 6 CC-chemokines. In animals without eosinophilia anti-Fas evoked no inflammatory response.
An efficient inducer of eosinophil apoptosis in airway tissues in vivo, anti-Fas mAb evoked unprecedented asthma-like inflammation in mouse allergic airways. This outcome may partly reflect the ability of anti-Fas to evoke direct cytolysis of non-apoptotic eosinophils in airway tissues. Additionally, since most apoptotic tissue eosinophils progressed into the pro-inflammatory cellular fate of secondary necrosis this may also explain the aggravated inflammation. Our data indicate that Fas receptor mediated eosinophil apoptosis in airway tissues in vivo may cause severe disease exacerbation due to direct cytolysis and secondary necrosis of eosinophils.
Apoptosis of inflammatory cells followed by their swift removal through phagocytosis is considered a major mechanism of resolution of inflammatory conditions [1, 2]. The most common chronic inflammatory disease, asthma is characterized by eosinophilia, epithelial derangement, plasma exudation, and hypersecretion [3, 4]. The role of the eosinophil in this disease is currently under intense investigation  and much interest has been devoted to apoptosis of eosinophil granulocytes [6, 7]. In the absence of growth factors or in the presence of glucocorticoids, eosinophils in vitro exhibit massive apoptosis and, eventually, secondary necrosis [8–10] occurs. A specific mode of inducing death through apoptosis is stimulation of Fas antigen (Fas), a cell surface protein expressed in most cells including eosinophil granulocytes . Fas may also trigger an alternative death pathway leading to cytolysis of cells without prior apoptosis . Eosinophil cytolysis causing extra-cellular spilling of eosinophil granules commonly occurs in asthmatic bronchi  but it is not known whether stimulation of the Fas-receptor may evoke cytolysis of eosinophils.
Apoptosis of eosinophil granulocytes is effectively induced in vitro by cross-linking of Fas membrane receptors with agonistic anti-Fas monoclonal antibody (mAb) [11, 14, 15]. Similarly, administration of anti-Fas mAb intra-nasally to the lungs of allergic mice has been shown to induce apoptosis of eosinophils in the airway lumen . This latter finding is of interest because apoptotic eosinophils have also been observed in asthmatic sputa following disease exacerbation . As a corollary it has been suggested that agents inducing eosinophil apoptosis may be developed as novel anti-asthma drugs [17–19]. However, the occurrence of apoptotic cells in the airway lumen cannot tell about the presence of such cells in the airway tissues . Indeed, apoptotic eosinophils have so far rarely have been seen in airway tissues  where eosinophils instead may be silently eliminated from the tissue through alternative clearance mechanisms such as egression into the airway lumen followed by mucociliary clearance [21, 22]. Even at resolution of established airway eosinophilia, spontaneously or by effects of anti-inflammatory steroids, apoptotic eosinophils have not been detected in lung tissues . The absence of apoptotic eosinophils in human diseased tissues together with the common occurrence of cytolytic eosinophils suggest that these cells are more prone to undergo cytolysis than apoptosis in inflamed airways (35). Also, since inducement of apoptosis in tissue eosinophils has not yet been compellingly demonstrated it remains speculative what actually may result in vivo when apoptosis of these cells occurs.
Differing from the prior reports, that focused on airway lumen data [7, 23], this study explores airway tissue effects of anti-Fas mAb given to mouse allergic airways with already established eosinophilic inflammation. Importantly, we have included a detailed transmission electron microscopy analysis to assess cell phenotypes such as apoptotic and cytolytic cells that are basically defined by ultrastructural characteristics . Here we demonstrate that anti-Fas mAb evoked apoptosis of more than 1/4th of the airway tissue eosinophils and that a majority of these cells proceeded to undergo secondary necrosis. Direct cytolysis of non-apoptotic tissue eosinophils was also induced by the present anti-Fas mAb treatment. Furthermore, at variance with previous interpretations of findings in the airway lumen and in vitro [7, 17, 23] we now demonstrate that the established allergic inflammation of airway-lung tissues was not resolved. On the contrary, as indicated by a wide range of indices, the allergic eosinophilic inflammation was greatly aggravated producing for the first time in mouse models several hallmarks of human asthma. This in vivo study thus demonstrates an unprecedented asthma-like histopathology in mouse airways and, unravels significant risks involved in drug-induced stimulation of death-receptors.
8–10 weeks old male C57BL/6 mice (Bomholtgard, Denmark) were used. Mice were kept in well-controlled animal housing facilities and fed ad libitum. The study was approved by the Regional Ethics Committee in Malmoe-Lund, Sweden.
Allergen sensitization and challenge protocol
After the lung tissue eosinophilia was established (day 22), 30 μg of anti-Fas mAb (purified hamster Anti-mouse Fas antibody, clone Jo2; Pharmingen, Palo Alto, CA) or a matched isotype control antibody (hamster IgG, control) was administered to the lungs via the intranasal route as previously described . The dose of anti-Fas mAb was chosen according to a dose-response study carried out by Tsuyuki et al where 30 μg was markedly effective at inducing apoptosis of airway luminal eosinophils . Unless otherwise stated each experimental group consisted of 8 animals. Outcome measurements were made at 8 and 24 h after each treatment (OVA/OVA+ IgG and OVA/OVA + anti-Fas mAb) (Figure 1). One group of animals with established eosinophilia treated with anti-Fas mAb was followed for 72 h after intra-nasal treatment (n = 3). Importantly, effects of anti-Fas mAb were also examined in mice lacking eosinophilic airway inflammation. Thus mice immunized with OVA and subjected to saline challenges were given anti-Fas mAb (or the isotype control IgG) and airway histopathology examined after 24 h. All animals were sacrificed by ip injection of pentobarbital immediately followed by bronchoalveolar lavage (BAL) and dissection of the lungs and tracheobronchial airways.
Bronchoalveolar lavage (BAL) and quantification of luminal cells
BAL was performed via a ligated tracheal cannula. One ml of PBS was allowed to passively enter the lungs at a pressure of 10 cm H20. This procedure was carried out twice. The obtained BAL-fluid (BALF) from each animal was immediately centrifuged (700 g, 5 min) and the supernatant frozen for ELISA analysis. The cell pellet was washed and resuspended in 250 μl PBS containing 10% FCS. The total number of cells was quantified using a hemocytometer and 5 × 105 cells cytocentrifuged to microscope slides. Differential cell counts were performed on May-Grünwald Giemsa stained slides and percentage of eosinophils, lymphocytes, neutrophils, and macrophages determined by counting 200 cells in a blinded manner. To obtain the absolute number of each leukocyte subtype in each BALF, the percentage of cells was multiplied by the total number of cells recovered from the BAL.
Lung tissue processing for histology analysis
From each animal 4 tissue samples were taken from the superior lung lobes at the level just below the root of the lung. One tissue sample was immersed in Stefanini's fixative (2% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer pH 7.2) overnight, rinsed repeatedly in Tyrode buffer supplemented with 10% sucrose, and finally frozen in TissueTEK (Miles, Inc., Elkhart, IN). The frozen specimens were stored at -80°C until used for histochemistry. A separate sample was immersed overnight in buffered 4% paraformaldehyde (pH 7.2) and thereafter dehydrated and embedded in paraffin. An additional sample was placed in a fixative consisting of a mixture of 3% formaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2 and used for transmission electron microscopic (TEM) analysis. The rest of the lung tissue was immediately frozen for mRNA analysis.
Staining and counting lung tissue eosinophils
Eosinophils were detected by histochemical visualization of cyanide-resistant eosinophil peroxidase (EPO) activity . In brief, 5 μm cryosections were incubated for 8 min at room temperature in PBS buffer (pH 7.4) supplemented with 3.3-diaminobenzidine tetrahydrochloride (60 mg / 100 ml; SIGMA), 30% H202 (0.3 ml / 100 ml), and NaCN (120 mg / 100 ml). Slides were then rinsed in tap water and mounted in Kaisers medium (Merck, Darmstadt, Germany). Eosinophils were identified by their dark brown reaction product and quantified as number of peribronchial eosinophils / 0.1 mm2 tissue area.
Staining of mucus-containing cells, mucus secretions, and mucus-plasma plugs
5 μm sections of paraffin embedded lung tissue were cut, dewaxed to water and then stained with periodic acid-Schiff reagent (PAS) as previously described . Epithelial integrity was examined and specific signs of injury-repair processes  were looked for. A mucus plug index was established as number of large and medium airways with tethered secretions/plugs divided by the total number of airways in each tissue section and multiplied by 100 to obtain percentage values. The presence of mucus plugs was also confirmed by transmission electron microscopy. Immunostaining for fibrinogen was performed using a polyclonal Ab (rabbit anti-fibrinogen 1:320, Dako, Copenhagen Denmark) and visualized using a secondary FITC antibody (swine anti-rabbit 1:80, Dako, Copenhagen, Denmark.).
Detection of apoptosis, secondary necrosis and eosinophil cytolysis
Apoptotic cells in the lung tissue were mainly detected using TUNEL-technique. A combined staining with TUNEL and Chromotrope-2R identified apoptotic eosinophils as previously described . Importantly, to assess an apoptotic morphology, detect engulfed eosinophils, and different activation grades of eosinophils, ultrastructural analysis using transmission electron microscopy was also performed as previously described . Ultrathin sections (60–80 nm) for electron microscopy were cut on an LKB MK III ultratome and contrasted with uranyl acetate and lead citrate. The sections were examined using a Philips CM-10 transmission electron microscope and the ultrastructural criteria for eosinophil apoptosis were eosinophils displaying cell shrinkage, intact cell membrane and nuclear chromatin condensation as previously described . Secondary necrosis was defined as cells exhibiting typical features of apoptosis e.g. nuclear condensation, but with clear signs of membrane rupture and extra cellular distribution of cell debris. Macrophages were identified using TEM and their content of eosinophil granules or whole eosinophil cell material was also quantified using TEM. Eosinophil cytolysis, which emerges as a major mode of eosinophil degranulation in asthma and rhinitis is characterized by chromatolysis of the cell nucleus and rupture of the cell membrane, whereby the protein-rich specific eosinophil granules are released into the tissue .
Measurement of mRNA expression
Total RNA from the lungs was extracted with RNAzol B (Tel-Test, Inc., Friendswood, TX) according to the manufacturer's protocol. Chemokine mRNA expression was determined by multiprobe RNAse protection assay (RPA) using the Riboquant RPA kit (mCK-5, Pharmingen, San Diego, CA), according to the supplier instructions and as previously described . The identity and quantity of each mRNA species in the original RNA sample were then determined based on the signal intensities given by the appropriately sized, protected probe fragment bands. 1 μg RNA was loaded for each sample and the differences in sample loading were normalized by a factor of the ratio of the housekeeping genes L32 and GAPDH.
Histology analyses were performed and quantified in a blinded manner. Tissue sections from eight animals were investigated in each treatment and control group. An Olympus BX60 microscope, equipped with an Olympus DP50 digital camera was used for imaging. Wilcoxon Rangsumtest for statistical analysis was performed using Analyze It™ (Analyse-it software, Ltd. Leeds, UK). Data are expressed as mean ± SEM. A value of p < 0.05 was considered statistically significant.
Fas-induced apoptosis and reduced number of eosinophils in the airway lumen
Fas-induced apoptosis of lung tissue eosinophils and insufficient clearance of apoptotic eosinophils
Viable, apoptotic, necrotic and cytolytic eosinophils in mice with OVA-induced established lung tissue eosinophilia.
Viable eosinophils A
OVA/control 8 h
3 ± 1
OVA/FAS 8 h
69 ± 7
19 ± 5
8 ± 3
4 ± 1
12 ± 4
OVA/control 24 h
5 ± 0.5
OVA/FAS 24 h
70 ± 4
13 ± 3
15 ± 2
2 ± 0.7
24 ± 2
Fas-induced secondary necrosis of apoptotic tissue eosinophils
Apoptotic eosinophils in the late stages of apoptosis, not being engulfed, proceeded to undergo secondary necrosis. Already at 8 h following treatment with anti-Fas almost half of the apoptotic eosinophils and at 24 h a majority of them exhibited secondary necrosis (Table 1). Typical signs of the secondary necrosis were a condensed dark nucleus and cell membrane rupture (Figures 4C and 5C) as previously described . These cells further exhibited piecemeal degranulation of the specific granules (Figure 5C).
Primary cytolysis of non-apoptotic tissue eosinophils
After treatment with anti-Fas mAb 2–4 % of the tissue eosinophils exhibited primary cytolysis more so at 8 h than at 24 h (Table 1). As described previously for eosinophils in human diseased airway tissues [13, 20, 29] these cells were without signs of apoptosis, exhibited little piecemeal degranulation and were characterized by chromatolysis and cell membrane rupture including the spilling of electron dense (protein-rich) specific granules into the tissue (Figure 5D, Table 1).
Anti-Fas mAb caused up-regulation of CC-chemokines
Additional signs of Fas-induced aggravation of airway inflammation
Administration of anti-Fas mAb to animals without eosinophilic inflammation
To investigate whether anti-Fas treatment produced inflammation in lungs where eosinophils were absent we used four groups of animals immunized with OVA and challenged with saline. In these animals, that did not develop eosinophilic inflammation and goblet cell metaplasia, neither anti-Fas mAb treatment nor isotype control treatment induced apoptosis (at 8 h and 24 h). Importantly, neutrophilia or other pro-inflammatory signs were not detected in the saline-challenged and anti-Fas treated lung tissues.
This study demonstrated that anti-Fas mAb induced eosinophil apoptosis in both airway lumen and tissue. However, this treatment did not resolve the established allergic eosinophilic inflammation. Instead, of being engulfed a majority of the apoptotic tissue eosinophils underwent secondary necrosis. Additionally, the Fas receptor stimulation evoked direct cytolysis of non-apoptotic tissue eosinophils. As a result we could demonstrate that an efficient inducer of eosinophil apoptosis in vivo, anti-Fas mAb produced unprecedented asthma-like inflammation involving degranulation of eosinophils, increased expression of CC-chemokines, epithelial derangement, plasma exudation, neutrophilia, tethered hypersecretion, and occurrence of significant mucus-plasma plugs in mouse allergic airways. Yet, anti-Fas treatment of mice without airway eosinophilia did not evoke any sign of inflammation. The present data on airway tissue events in vivo contradict the current notion, based on interpretations of findings in vitro and in the airway lumen, that inducement of eosinophil apoptosis is a therapeutic modality in asthma.
Current allergic mouse models of asthma are characterized by eosinophilia and by airway remodeling including transformation of the epithelium into a secretory (PAS-positive) cell lining . Otherwise the signs of inflammation are mild or absent, and the mouse models of asthma may be criticized for their lack of disease-like pathophysiology and histopathology . It is, therefore, of interest that the present mouse lungs could exhibit 'asthma-like' exudative, eosinophilic, epithelial, and neutrophilic inflammation. After the combined allergen challenge and anti-Fas treatment the present tissue eosinophils further showed several signs of activation ranging from cytolysis of non-apoptotic eosinophils to secondary necrosis of apoptotic eosinophils. Cytolysis has not previously been observed in mouse airway tissue eosinophils , but is of particular interest because this mode of degranulation is prominent in human airway eosinophilic diseases [29, 35]. Although the mechanisms involved in the present primary cytolysis of eosinophils are not known it is possible that Fas receptor stimulation directly induced cytolysis in these eosinophils through subcellular effects critically deviating from the apoptosis pathway . The time course of cytolysis in this study with more cytolysis occurring at 8 h than at 24 h (contrary to secondary necrosis) supports the possibility that Fas directly evoked this mechanism. Fas administration in this study also evoked tethered luminal secretions and mucus plugs, as observed in asthma , considerably more so than observed after allergen challenge alone (this study). Moreover, as indicated by fibrinogen immunoreactivity, the present anti-Fas treatment evoked a plasma exudation process that has not previously been demonstrated in the allergic mouse models of asthma. In allergic asthma luminal levels of a large protein such as fibrinogen may better than albumin reflect the plasma exudation process, a hallmark of the disease especially at exacerbations . Prior work has already established that anti-Fas antibody may evoke inflammatory responses such as hepatitis and pneumonitis . Indeed, the pro-inflammatory aspect of Fas has already prompted repeated studies on the effect of this agent in the airways of allergic mice . However, all previous workers, focusing largely on lumen findings, have arrived at the consensus notion that Fas-induced effects in mouse allergic airways are of an anti-inflammatory nature. The present study demonstrates the opposite. Our findings further indicate that careful studies are warranted to assess risks associated with treatment with adenovirus expressing Fas ligand. The latter construct was recently promoted as an anti-asthma treatment option .
Few non-eosinophilic cells became apoptotic in this study. We also detected no inflammatory response in anti-Fas-treated mice that had not developed lung eosinophilia indicating that eosinophlis were essentially involved in the present Fas-induced aggravation of allergic inflammation. Further studies are warranted to explain the present finding that both airway tissue and lumen eosinophils are particularly sensitive to Fas-receptor stimulation. The present observation that macrophages (or other cells) are not immediately engulfing the apoptotic eosinophils tallies with observations suggesting that pulmonary macrophages may be less efficient than commonly studied peritoneal macrophages as regards engulfment of apoptotic cells . Reflecting an insufficient clearance, necrosis of tissue-dwelling, apoptotic eosinophils was a prominent feature in this study. Secondary necrosis of apoptotic cells is a pro-inflammatory event  and so is likely the primary cytolysis of eosinophils . Both modes of cell death could thus be causally involved in the present aggravation of the allergic inflammation. In view of the dependence of Fas-induced inflammation on established lung eosinophilia the necrosis and the cytolysis phenomena may be major pathogenic mechanisms in this study. Also, as suggested by the present observations 3 days after anti-Fas treatment the induced inflammatory process is self-sustained continuing for a considerable length of time even in the absence of further airway provocations.
If cell clearance through apoptosis and engulfment are not working well in the lung other modes for non-inflammatory resolution of lung eosinophilia are needed. In fact, it was recently proposed that transepithelial cell egression may efficiently, and without affecting the integrity of the epithelial lining, resolve an established airway tissue eosinophilia . Egression into the airway lumen clearly occurred in this study as evidenced by a developing BAL fluid eosinophilia in allergen challenged animals. The occurrence of eosinophil apoptosis in the present Fas-exposed airway tissue must have reduced the egression simply because dying cells cannot migrate. Inferentially, failure of tissue eosinophils to egress into the airway lumen (due to their apoptotic condition) explains in part the present maintained tissue eosinophilia. A reduced egression would also explain in part the demonstration in this and previous studies  of anti-Fas mAb-induced diminution of lumen eosinophilia in allergic airway inflammation. The possibility of such complex relationships between cell numbers and phenotypes of airway lumen and tissue, respectively, underscores the difficulty in drawing conclusions merely based on airway lumen data. Similarly, it is puzzling that mouse eosinophils exhibit piecemeal dagranulation in the airway lumen  but not in the airway tissue [41, 42].
The present anti-Fas-induced inflammation was probably associated with de novo recruitment of eosinophils because the expression of several eosinophil-recruiting chemokines , including eotaxin, and MIP-1α were markedly up-regulated. The present non-specific increase in CC-chemokines may in part be due to an unrestricted release of bioactive proteins from eosinophils undergoing cytolysis or secondary necrosis in airway tissues. Besides eosinophilia increased airway neutrophilia is common especially in severe asthma . The increased presence of neutrophils is therefore an interesting feature of the present anti-Fas treated airways. Fas itself may act as a chemoattractant for neutrophils  but neutrophilia was not detected in the present control group challenged with saline and receiving anti-Fas treatment. It has been suggested that tissue-toxic agents from neutrophils as well as eosinophils have a causative role in epithelial derangement. Epithelial cell loss alone may also evoke significant local neutrophilia in vivo . These aspects indicate that neutrophilia is an expected component in the present airways where degranulated eosinophils and epithelial derangement were prominent features.
This study demonstrates for the first time that eosinophil apoptosis can be induced to a great extent in airway tissues in vivo. But then, in contrast to current notions, this response was not associated with resolution of the established eosinophilic inflammation. This finding alone questions the proposed role of apoptosis for efficient, non-inflammatory clearance of a major granulocyte in allergic airways. Yet, we additionally discovered here that the combined effect of allergic eosinophilic inflammation and anti-Fas evoked not only apoptosis but also secondary necrosis as well as primary cytolysis of the tissue-dwelling eosinophils. These latter effects were associated with multifaceted inflammatory processes developing far beyond the allergic inflammation in this species and involving significant components of hallmark features of asthma. Hence, Fas-receptor stimulation of airway tissue eosinophils in vivo emerges as a significant pathogenetic mechanism and unravels significant risks involved in drug-induced stimulation of death-receptors.
This study was supported by the Medical Faculty, Lund University, Sweden, The Swedish Medical Research Council, The Heart and Lung Foundation Sweden. The authors thank Dr Eric Carleman and Britt-Marie Nilsson at the Electron Microscopy Unit, Lund University.
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