Eosinophils and lymphocytes are pathogenically important in allergic inflammation and sensitive to Fas-mediated apoptosis. Fas ligand (FasL) activity therefore should play a role in regulating the allergic immune response. We aimed to characterize the role of FasL expression in airway eosinophilia in Aspergillus fumigatus (Af)-induced sensitization and to determine whether FasL neutralization alters the inflammatory response.
Sensitized Balb/c mice were killed before (day 0) and 1, 7 and 10 days after a single intranasal challenge with Af. Animals received either neutralizing antibody to FasL (clone MFL4) or irrelevant hamster IgG via intraperitoneal injection on days −1 and 5. FasL expression, BAL and tissue inflammatory cell and cytokine profile, and apoptosis were assessed.
Postchallenge FasL gene expression in BAL cells and TUNEL positivity in the airways coincided with the height of inflammatory cell influx on day 1, while soluble FasL protein was released on day 7, preceding resolution of the inflammatory changes. Although eosinophil numbers showed a negative correlation with soluble FasL levels in the airways, MBP+ eosinophils remained TUNEL negative in the submucosal tissue, throughout the 10-day period after Af challenge. Systemic FasL neutralization significantly enhanced BAL and tissue eosinophil counts. This effect was associated with increased activation of T cells and release of IL-5, IL-9, and GM-CSF in the BAL fluid of mice, indicating an involvement of pro-eosinophilic survival pathways.
FasL activity may play an active role in resolving eosinophilic inflammation through regulating T cells and pro-eosinophilic cytokine release during the allergic airway response.
Engagement of a Fas-expressing target cell by a Fas ligand (FasL)-bearing effector can result in the programmed cell death of the target. Oligomerization of the Fas receptor by FasL with subsequent recruitment of adapter molecules and activation of the caspase cascade ultimately results in mitochondrial destabilization and destruction of substrates essential for cell survival [1-6]. Translation of these in vitro observations to human asthma is an emerging area. Airway inflammation in asthma is now well described , and its clinical importance is underscored by the fact that regular use of anti-inflammatory agents (particularly corticosteroids) improves symptoms and exacerbations, whereas monotherapy with bronchodilators does not [8, 9]. Many asthmatics have persistent symptoms and residual inflammation despite these drugs, pointing to abnormal activation and/or impaired clearance of inflammatory cells . FasL-sensitive macrophages, lymphocytes, and eosinophilic and neutrophilic granulocytes figure prominently in the pathogenesis of asthma. The theory that Fas-mediated elimination of inflammatory cells should be an important means of modulating airways inflammation is not new but has been difficult to prove in the context of a relevant disease process. [11, 12].
There is increasing evidence that Fas/FasL interactions lead to multiple different pathways involved in regulation of immune and inflammatory cell functions. Our study characterizes the time-dependent FasL gene and protein expression in the lung in a murine asthma model [13, 14] and provides evidence that systemic neutralization of endogenous FasL activity prolongs airway eosinophilia, supporting a role for Fas/FasL interaction in resolving allergic airway inflammation.
Materials and methods
Aspergillus fumigatus (Af) sensitization and challenge model
Six- to 8-week-old female BALB/c mice (weighing approximately 20 g) were sensitized and challenged (Fig. 1A) as previously described [13, 14]. FasL was neutralized by intraperitoneal injections with anti-FasL (MFL4 provided by Dr. Hideo Yagita, Juntendo University, Tokyo, Japan)  in PBS (Fig. 3A) or irrelevant hamster IgG (Jackson ImmunoResearch, West Grove, PA, USA). Protocols were approved by the Institutional Animal Care and Use Committee.
Bronchoalveolar lavage (BAL) and tissue collection
BAL was obtained and lungs were collected in paraformaldehyde or RNAlater® as described previously [13, 14, 16]. The BAL fluid was processed for total and differential cell counts, and the cell-free supernatant (stored at −80°C) was analyzed for soluble Fas and FasL levels (R&D Systems, Minneapolis, MN, USA). Cytokines were measured by the Q-Plex™ mouse inflammation cytokine array (Biolegend, San Diego, CA, USA).
Paraffin-embedded, ethanol-fixed tissue sections were studied for the presence of apoptotic cells (TUNEL assay; Roche Applied Science, Indianapolis, IN, USA; Fig. 1E), tissue eosinophils (anti-mouse major basic protein [MBP] antibody provided by Dr. Jamie Lee; Mayo Clinic, Scottsdale, AZ; Figs 1E and 4E), and FasL expression (polyclonal anti-FasL N20 antibody, Fig. 2C/a–d, or irrelevant rabbit IgG, Santa Cruz Biotechnology, Santa Cruz, CA, USA; Fig. 2C/g).
FasL expression by BAL cells was studied by immunocytochemistry in acetone/methanol-fixed cytospins using N20 labeling (Fig. 2B) and by indirect immunofluorescence in live cells using biotinylated anti-FasL MFL4 and MFL3 clones (not shown). The specificity of lung FasL staining was confirmed in OCT-embedded (Tissue-Tek®; Fisher Scientific, Pittsburgh, PA, USA), snap-frozen blocks of lung tissue processed as previously described  and labeled with anti-FasL MFL3 (not shown) or MFL4 (Fig. 2C/f).
TUNEL+ cells were individually quantitated (NIS-elements® image analysis software, Nikon Inc., Melville, NY, USA) by blinded observers. The density threshold was set to trace all positive cells within the subadjacent peribronchial tissue for a given length of airway. TUNEL results were corroborated with a PE-conjugated activated caspase 3 antibody (BD Pharmingen, San Jose, CA, USA; data not shown).
Peribronchial MPB+ eosinophils were assessed the same way (Fig. 4E/b), but because eosinophils were occasionally crowded and touching in areas of intense inflammation and border-finding algorithms for individual cells failed there, we divided the total cellular MPB+ area for a given peribronchiolar region by the average area of a single eosinophil (53 μM2; a constant obtained through direct measurement of 20 individual, well-defined cells). It should be noted that the MBP+ area analyzed was always overtly cellular by direct inspection and not related to extracellular deposition. Tiny (<40 μM2) objects were excluded from this MBP+ area as probable artifacts.
First-strand cDNAs were synthesized from 5 μg of total RNA using random primers and the High Capacity cDNA synthesis kit (Applied Biosystems, Foster City, CA, USA). Quantitative real-time PCR was performed using the TaqMan® gene expression assay for murine FasL (mRNA from Applied Biosystems) normalized to GAPDH mRNA.
Splenic lymphocyte function
Splenocytes were isolated, washed, and cultured at 8 × 106 cells/ml in U-bottom 96-well plates (Fisher Scientific) in RPMI 1640 medium; 3H-thymidine uptake was assessed as previously described .
Results are expressed as mean ± standard error of the mean (SEM). Time courses and dose responses were compared by two-way anova. Individual comparisons were made by the nonparametric Mann–Whitney U test. Regression analysis was performed using the Spearman's correlation test. Statistical significance was set at a P-value <0.05. Data were analyzed using Microsoft Excel and GraphPad Prism 5 for Windows.
The peak TUNEL positivity coincided with inflammatory influx in the airways, but eosinophils remained TUNEL negative after Af challenge
We previously showed that prevention of the inflammatory resolution after allergen inhalation was associated with decreased cell death of MBP+ eosinophils . To investigate the role of eosinophil apoptosis, here we studied the time-dependent relationship between airway eosinophil numbers and TUNEL positivity after Af inhalation in sensitized BALB/c mice (Fig. 1A). The numbers of inflammatory cells were significantly increased with an eosinophil predominance on day 1 (Fig. 1B) and a subsequent return to near baseline by day 10 (Fig. 1C). Pro-eosinophilic (IL-4, IL-5, GM-CSF and IL-9) cytokine release paralleled the height of inflammation, and these cytokines (except IL-9) positively correlated with BAL eosinophil numbers on day 1 after Af challenge (Fig. 1D).
TUNEL+ apoptotic and aponecrotic cells (green) were frequently present postallergen challenge (a representative day 1 photomicrograph is shown, Fig. 1E/d). The greatest numbers of TUNEL+ cells paralleled the peak of eosinophilia on day 1 after Af challenge (Fig. 1F). However, we found only occasional MBP+ cell (red) co-localization with TUNEL+ nuclei, while most MBP+ cells remained TUNEL- (Fig. 1E/d,G). Thus, TUNEL positivity coincided with the height of inflammation, but apoptosis was not associated with resolution of the Af-induced airway response.
Soluble FasL release into the airways negatively correlated with eosinophil cell numbers
To study the relationship between FasL expression and airway eosinophil numbers, we investigated FasL mRNA and protein levels in BAL cells, supernatant and lung tissue after Af challenge of sensitized mice. In the BAL cellular compartment, cytoplasmic/membrane protein expression corresponded with increased mRNA activation on day 1 (Fig. 2A,B). Based on nuclear morphology, the FasL-positive cells appeared mononuclear, while polymorphonuclear cells and cells with eosinophil-like nuclei remained FasL negative (Fig. 2B, a representative day 1 sample). In the lung tissue, expression of FasL peaked in the proximal airway epithelial cells on day 7 postchallenge (Fig. 2C). Although we were not able to find commensurate increases in lung tissue FasL mRNA by microarray (data not shown), de novo FasL synthesis was previously demonstrated by studies in human airway epithelial cells in vitro and in Balb/c mice in vivo, indicating airway epithelium as the source of FasL. Local release (BAL vs serum) of soluble FasL indeed corresponded with the peak epithelial expression on day 7 (Fig. 2D) and showed a significant negative correlation with the percentage of BAL eosinophils post-Af challenge (r = −0.6; P < 0.01; Fig. 2E). Meanwhile, levels of soluble Fas in the cell-free BAL supernatant increased only on day 1 (from 247 ± 12 to 407 ± 53 pg/ml) before returning to (and remaining at) near baseline on days 7 and 10.
Systemic FasL neutralization enhanced lymphocyte activation and cytokine release after Af challenge
In the asthmatic airways, many factors can regulate survival/death of eosinophils including cytokines released by the highly FasL-sensitive T cells [20-23]. To study how systemic blockade of FasL affects T-cell function, we treated mice with the neutralizing anti-FasL antibody MFL4 or control hamster IgG by intraperitoneal injection (Fig. 3A). MFL4 enhanced proliferation of splenic T cells (Fig. 3B,C) harvested on day 1 or 7, (but not on day 10 or in nonchallenged [day 0] mice) both at baseline (left panels) and after PHA stimulation (right panels). Interestingly, dexamethasone responsiveness of the T cells was partially reversed in the MFL4-treated groups (Fig. 3C), suggesting that FasL blockade induced heightened activation and a relative T-cell glucocorticoid resistance.
MFL4 also enhanced release of the T-cell cytokines IL-5, IL-9, and GM-CSF in the lung in a time- and dose-dependent manner. Figure 3D shows cytokine levels harvested on day 1 post-Af challenge (BAL cytokine release was negligible at other time points). Levels of soluble Fas ligand negatively correlated with IL-5, IL-9, and GM-CSF recovered from the BAL fluid of MFL-treated mice on day 1, suggesting that systemic FasL neutralization significantly enhances T-cell function and local release of pro-eosinophilic (“survival”) cytokines during the Af-induced inflammatory response.
The BAL and lung tissue inflammatory cell profile was assessed to investigate whether the increased T-cell-derived pro-eosinophilic cytokine release was associated with altered eosinophil count after MFL4 treatment. Compared to the PBS-treated mice (0 mg), the greatest, dose-dependent MFL4 effects on the total BAL cell count were seen on day 7 post-Af challenge (Fig. 4A) with increased numbers of macrophages and eosinophils (Fig. 4B,D) and decreased soluble FasL levels (Fig. 4C). MBP+ tissue eosinophils (Fig. 4E/a) counted in the peribronchial region using density threshold morphometry (Fig. 4E/b) showed significantly elevated numbers after Af challenge in the MFL4-treated mice compared with the control antibody-treated mice (P < 0.05, two-way anova, Fig. 4F). Taken together, blockade of Fas/FasL interaction enhanced airway inflammation and delayed eosinophil clearance after allergen challenge.
This study demonstrates that resolution of airway eosinophilia was not directly associated with apoptosis or FasL expression by these cells in allergen-exposed mice. Soluble FasL release from the airway epithelium (that peaked 7 days after Af challenge) was negatively proportionate with eosinophil counts and pro-eosinophilic cytokine levels in the airways. Systemic FasL blockade increased T-cell activation and dose- and time-dependently enhanced IL-5, IL-9, GM-CSF, and airway eosinophilia. We conclude that FasL-mediated pathways are important in attenuating eosinophilic airway inflammation possibly via inhibition of survival cytokine production.
The initial interest in Fas/FasL interactions in major asthma effector cell types was piqued in the mid-1990s when ligand-mediated apoptosis was demonstrated in eosinophils  and T cells . In a model of ozone-induced enhancement of airway eosinophilia, we previously found association with diminished cell death and decreased FasL expression in the lung of allergen-exposed mice . Other studies exogenously administered FasL activity into the airways of mice either by agonist antibody or by viral expression consistently induced a decline in BAL eosinophil numbers postchallenge but had variable effects on tissue infiltration and granulocyte apoptosis [24-27]. To investigate the direct relationship between FasL expression, cell death, and resolution of airway eosinophilia, in the current study we applied a different approach from previous investigations, in that endogenously derived FasL activity was neutralized during the allergen-induced airway inflammatory response . Studies using the functional Fas (lpr)-deficient  and FasL (gld)-deficient  mouse models were criticized for their tendency toward immune dysregulation and frank autoimmunity that can confound interpretation of inflammatory endpoints . In this respect, our extensively characterized anti-FasL approach  provides a system free of confounding immune/inflammatory pathologies. The increased airway eosinophilia seen in the allergen-challenged mice after FasL neutralization in this model could be due to several possible pathways.
One would intuit that blocking FasL should enhance inflammation by preventing apoptosis of Fas+ targets. However, we found no inhibition of apoptosis in MFL4 treated mice. Peak inflammatory changes in the mice were characterized by the major presence of abnormal TUNEL+ cells, suggesting secondary necrosis (i.e. aponecrosis)  and supporting previous observations that inflammatory cells at sites of allergic inflammation may be cleared by means other than classical apoptosis [27, 31]. The presence of a negligible number of TUNEL+/MBP+ cells in the lung tissue of allergen-challenged mice also suggested that apoptosis may not have been the main pathway of eosinophil clearance in this model.
After Af challenge, FasL mRNA activation paralleled inflammatory cell influx in the BAL, but overtly positive cell surface expression was rare and showed a mononuclear rather than eosinophil morphology. Based on our results, we speculate that the pro-inflammatory MFL4 effects were likely mediated through inhibition of soluble, rather than membrane-bound, FasL in the lung. These forms have markedly different effects on cell function: While only the membrane-bound form can induce apoptosis, soluble FasL has nonapoptotic roles promoting autoimmunity, tumourigenesis, and immunosuppression . Soluble FasL (thought to be cleaved by matrix metalloproteinases in the BAL supernatant ) peaked in the BAL fluid on day 7, while serum levels remained low throughout after Af challenge. Using several anti-FasL reagents, we confirmed previous reports [18, 19, 34] showing bronchial epithelial cell expression of FasL immunoreactivity, which was also strongly increased on day 7. Thus, the predominant source of soluble FasL after allergen challenge is the airway epithelium, and a time-dependent FasL release coincides with resolution of inflammation.
The increased production of Th2-type cytokines and heightened T-cell proliferation from the MFL4-treated mice strongly suggest that FasL acts by limiting the expansion of antigen-specific T cells, a mechanism that would be consistent with known functions of Fas/FasL interactions. Given the strong CD4 T-cell dependence of the Af sensitization model , one way to demonstrate whether FasL inhibition is acting via T cells would be through adoptive transfer experiments. Such experiments have been performed in a S. mansoni-induced asthma model using Fas-deficient (lpr) T cells  as well as FasL-deficient (gld) T cells . Both studies supported that delayed resolution of eosinophilia is a downstream effect of Fas deficiency on T cells, not eosinophils. Further, our study as well as the models using the lpr[22, 28] or the gld mice  points to the significance of T-cell-derived survival cytokines in enhancing allergen-induced airway eosinophilia in the absence of Fas/FasL activity. We proposed in a previous study  that pro-inflammatory cytokines IL-5 and GM-CSF in addition to rendering eosinophils resistant to Fas/FasL-induced apoptosis, may also inhibit FasL expression. This is now refuted in this current study because increased levels of the pro-eosinophilic IL-5, IL-9, and GM-CSF in the BAL fluid of mice coincided with the peak of BAL cell mRNA expression for FasL. The fact that FasL neutralization resulted in significantly increased expression of these cytokines would suggest instead a reverse regulatory pathway in which FasL activation exerts an inhibitory effect on pro-inflammatory cytokine release during the allergic airway response.
In summary, the enhanced and prolonged airway eosinophilia after systemic FasL neutralization provides mechanistic support that endogenously released FasL participates in resolution of airway inflammation. The inverse relationship between FasL protein expression, T-cell activation, and pro-eosinophilic cytokine production highlights the importance of these Fas-sensitive pathways in keeping allergic airway inflammation at check.
This study was supported by NIH grants R01HL076646 (JZ & AH), 1RC1ES018505 (AH), and R01AI072197 (AH).
Satish K. Sharma, PhD, performed the immunohistochemistry, ELISA, and qPCR studies, assembled database for the study, analyzed data, and participated in manuscript preparation. Francisco A. Almeida, MD, participated in experimental design and mouse model of allergic airway sensitization. Sonja Kierstein, PhD, participated in experimental design, mouse models of allergic airway sensitization, and manuscript preparation. Laszlo Hortobagyi performed the splenocyte proliferation studies. Timothy Lin, MD, and Allyson Larkin, MD, participated in the mouse model of airway sensitization. Jonathan Peterson participated in the immunocytochemistry studies and the mouse model of airway sensitization. Hideo Yagita, PhD, developed the monoclonal anti-FasL antibodies used in this study and provided advice and guidance for the evaluation of their effects. James G. Zangrilli, MD, and Angela Haczku, MD, PhD, designed the study, analyzed data, and prepared the manuscript.
Conflict of interest
James G. Zangrilli MD: Currently employed by Astra-Zeneca; other authors have no conflict of interest.