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.