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 (11
) and T cells (12
). 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 (16
). In 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 post challenge, but had variable effects on tissue infiltration and granulocyte apoptosis (24
). 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 (15
). Studies using the functional Fas (lpr
) and FasL (gld
) deficient mouse models were criticized for their tendency towards immune dysregulation and frank autoimmunity that can confound interpretation of inflammatory endpoints (29
). In this respect our extensively characterized anti-FasL approach (15
) 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 a major presence of abnormal TUNEL+ cells suggesting secondary necrosis (i.e. aponecrosis) (30
) and supporting previous observations that inflammatory cells at sites of allergic inflammation may be cleared by means other than classical apoptosis (27
). Presence of a negligible number of TUNEL+
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.
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 non-apoptotic roles promoting autoimmunity, tumourigenesis and immunosuppression (32
). Soluble FasL (thought to be cleaved by matrix metalloproteinases in the BAL supernatant (33
)) 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
) showing bronchial epithelial cell expression of FasL immunoreactivity, that 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 (35
), 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 (22
) as well as FasL deficient (gld
) T cells (23
). 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
) or the gld
) point 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 (16
) that proinflammatory cytokines IL-5, GM-CSF, in addition to render eosinophils resistant to Fas/FasL-induced apoptosis may also inhibit FasL expression. This is now refuted in this current study since 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 proinflammatory cytokine release during the allergic airway response.
In summary, the enhanced and prolonged airway eosinophila 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.