Why asthmatics fail to resolve inflammation in their airways remains one of the unsolved problems in asthma (1
). Persistence of T cells and eosinophils in bronchial biopsies was found in persistent asthma and even in intermittent asthma (4
). These findings led us to question how Th2 inflammatory responses normally resolve in murine models of asthma. The objective of this investigation was to determine the potential role of the death receptor, Fas (CD95), in antigen-induced Th2-mediated airway inflammation in mice. We find that Fas+
T cells play a pivotal role in the resolution of airway inflammation, mucus production, and AHR. These findings in mice may shed light on potential mechanisms of asthma pathogenesis.
Several studies have suggested that dysregulated apoptosis of eosinophils may be involved in human asthma. Eosinophils express the Fas receptor and undergo apoptosis in vitro and in vivo when bound by FasL or an agonizing antibody (30
). However, eosinophils from asthmatic lungs undergo less apoptosis than normal controls and express more Bcl-2, and treatment of human asthma with corticosteroids leads to an increase in the number of apoptotic eosinophils (33
). In fact, asthma severity has been directly correlated to reduced eosinophil apoptosis in induced-sputum samples (35
). Yet, how eosinophil apoptosis is regulated in asthma remains undefined.
In our study, we demonstrated that Fas-deficient Th2 cells were sufficient to drive the extended course of airway inflammation in sensitized and challenged Lpr mice. These data suggest that the fate of eosinophils in the lungs and airways is largely dependent on T cells. Our results do not discount the effect of eosinophil apoptosis mediated by Fas or other mechanisms. Furthermore, because we have not examined eosinophil apoptosis directly in our model, we cannot exclude the possibilities that the lack of resolution is the result of lack of clearance of eosinophils or to enhanced or persistent recruitment of cells. Nevertheless, our findings suggest that resolution of airway eosinophilia inflammation is largely downstream of Fas expression on T cells. In human asthma, there were several reports that T cells also undergo less apoptosis. One report found that mitogen-stimulated peripheral blood T cells from asthmatic subjects failed to undergo the same degree of Fas-mediated apoptosis as T cells from the normal control subjects, thereby providing direct evidence for a defect in programmed cell death in the pathogenesis of asthma (5
). Additionally, defective expression of Fas messenger RNA and Fas receptor was found on pulmonary T cells from patients with asthma (36
). De Rose et al. reported that the defect in IFNγ production involved in the allergic immune response may be responsible for a decrease in apoptosis of allergen-activated T lymphocytes in the airways of atopic asthmatic patients (37
). Thus, our results in this new model have direct correlations with the findings of these studies in human asthma. In addition to dysfunctional Fas pathway on T cells, markedly reduced Fas ligand mRNA and protein in the airway epithelium during allergic airway inflammation induced by OVA in mice may be also involved in the pathogenesis of certain inflammatory conditions of the airway (38
Persistent Th2 inflammation has been one of the most difficult aspects of authentic asthma to model in animals. Many models for persistent inflammation require continued antigen exposure for long periods of time. When antigen exposure is ended, airway remodeling and AHR can remain for 3–4 wk; however, inflammation resolves quickly after the last antigen challenge (39
). Recently, there has been the development of other models that use either mite allergens (42
) or conventional OVA allergen with new technology for aerosol delivery (40
). However, all of these models require 20–40 nebulizations after one to two immunizations. Mice with a targeted deletion of the T-bet gene spontaneously demonstrated multiple physiological and inflammatory features characteristic of asthma (43
). Transgenic mice that overexpress IL-5 (44
), IL-9 (45
), IL-11 (46
), and IL-13 (47
) showed AHR, eosinophilic inflammatory response, and collagen deposition in the airways, indicating that chronic exposure to Th2 cytokines could also induce airway remodeling. Airway inflammation in these animal models occurs spontaneously without any antigen exposure. Thus, current methods for inducing chronic airway inflammation fall into two categories: those models in which the mice are repeatedly challenged for weeks or even months, and models of genetically manipulated mice that develop a spontaneous Th2-type airway inflammation in the absence of allergen exposure.
In this study, we now present data on a third method of inducing persistent airway inflammation. This novel model is not spontaneous, but allergen-induced, yet only requires two challenges after a single sensitization. Although it has been proposed that the persistent inflammation found in asymptomatic asthmatics is the result of chronic low level exposure to allergens, it is just as likely that the persistent inflammation in asymptomatic asthmatics is the result of defects in resolution of airway inflammation. Thus, the Fas-deficient T cell mice represent an altogether new model of persistent airway inflammation that does not require continuous challenges to induce this key feature of asthma.
Recently, Rajewsky et al. published their findings on ablation of Fas specifically in the T cell compartment (28
). They reported that between 8 and 12 mo, these mice develop severe pulmonary fibrosis and accumulation of inflammatory cells. Although T cell–specific Fas−/−
mice and our adoptively transferred Lpr>Rag−/−
mice are both Fas deficient on only T cells, the timing and type of lung pathology differs in the two models. Nevertheless, to test whether our findings could be explained by spontaneous lung disease, we adoptively transferred B6 and Lpr T cells into Rag−/−
mice but did not sensitize and challenge these animals. After 8 wk, no evidence of inflammation could be found (). Thus, it is highly unlikely that persistence of Th2-type inflammation in our model is related to the pulmonary fibrosis and inflammation that develops at a much later stage of life in T cell specific Fas−/−
Our study is supported by previous work from Gelfand et al., who demonstrated that AHR is increased in OVA-sensitized and challenged Fas-deficient mice compared with wild type at day 4 after the last challenge and that blockade of inflammation with anti–IL-5 treatment attenuates the response (18
). Interestingly, unlike our findings, these investigators did not find a delay in the resolution of inflammation in the Lpr mice. More importantly, in our adoptive transfer model, we now demonstrate that Fas deficiency specifically on T cells is sufficient to prolong inflammation despite normal Fas expression on eosinophils.
To investigate the possible mechanisms involved in the development of persistent airway inflammation in sensitized and challenged Lpr>Rag−/−
mice, T cell function was studied. We found that lung T cells from B6>Rag−/−
mice produced more IFNγ+
cells than lung T cells from Lpr>Rag−/−
mice and, interestingly, that the IFNγ-producing cells in the lungs were mostly CD4+
T cells. To test the hypothesis that development of IFNγ+
T cells may be a key mechanism involving timely resolution, IFNγ−/−
T cells were transferred to Rag−/−
mice. We found that IFNγ−/−
mice have a delayed resolution of eosinophilia and lung peribronchial perivascular inflammation (). These data suggest that the failure of Lpr T cells to produce IFNγ in the Lpr>Rag−/−
mice may play an important role in their inability to resolve their Th2-mediated inflammation. These results were also consistent with previous reports (21
) in which Th1 T cells inhibited Th2-induced eosinophilia and mucus production. Why effective antigen-specific Th1 cells fail to develop in the Lpr>Rag−/−
mice remains unclear. In fact, several reports suggest that Fas deficiency leads to greater Th1 responses (for review see reference 12
). Thus, further studies will be required to resolve these apparent contradictory studies.
There has been much controversy over the role of Th1 cells in asthma (for review see reference 1
). Although some studies have suggested a protective role for Th1 cytokines in allergy and asthma (50
), other investigators have suggested that Th1 cells enhance pulmonary inflammatory responses and AHR (52
). In our model, the data clearly demonstrate that Th2 cells play a stimulating role in the early phase of the airway inflammation, but that Th1 cells play an inhibiting role in the chronic phase of airway inflammation. Nevertheless, the development of our novel animal model of asthma provides a new opportunity to study the mechanisms involved in chronic Th2 inflammation as well as the pathological outcomes of this long-term inflammation. Further investigations in the role of Fas in regulating Th2 immunity may translate into better treatments for asthma and other Th2-mediated diseases.