Recent studies have established that CD4 Th2 cells and their cytokines initiate an inflammatory response in the respiratory tract with many features of asthma. In contrast, Th1 cells lead to inflammation but exhibit none of the asthmatic pathology. As both Th1 and Th2 cells have been identified in the lungs of asthmatic patients, we investigated whether Th1 cells could regulate allergic airway pathology. In this report, we show that coactivation of Th1 and Th2 cells in the lung leads to a dominance of Th1 effects, inhibiting both airway eosinophilia and mucus production. Th1 cells, through the production of IFN-γ, inhibit these Th2-induced effects, not by regulating Th2 cell activity, as previously suggested, but by blocking downstream pathways induced by Th2 cytokines. Furthermore, the marked inhibitory effects of Th1 cells occur without an increase in airway inflammation. Thus, it appears that Th1 cells block critical pathologic changes that contribute significantly to morbidity and mortality in asthma. Our data show that IFN-γ inhibits airway eosinophilia even during polarized Th2-type responses, indicating that Th1 cells in the airways of asthmatics may be active in controlling disease.
In addition, these studies are the first to show that the development of inflammatory pathology in asthma can be differentially controlled. Whereas airway eosinophilia depends on the presence of activated Th2 cells in the lung, mucus can be induced by different types of inflammatory infiltrates as long as IFN-γR signaling is blocked. Th1 cells fail to stimulate mucus because IFN-γ inhibits its production. Th1 cells do not induce eosinophilia, most likely due to a lack of IL-5 31
Th1 responses have been proposed to protect against asthma. This theory is based on Th2-dominant lymphocyte populations in the airways of asthmatics and evidence that Th1 responses protect in asthmatics and in animal models of asthma 27111213
. Th1 cells may inhibit Th2 cell function at different stages in the effector response. Th1 cells, through the production of IFN-γ, have been shown to inhibit Th2 cell cytokine production and Th2 cell proliferation in vitro 3233
. In mice, the Th1 cytokine IFN-γ has inhibitory effects on Th2-induced airway eosinophilia and AHR. When administered before inhaled antigen challenge, IFN-γ reduced the number of CD4 T cells in the respiratory tract 262829
or reduced Th2 cytokine secretion 2634
. These effects may result from inhibition of Th2 cell recruitment by IFN-γ. Once Th2 cells are present in the respiratory tract, IFN-γ suppresses the resolution of Th2-induced inflammation, as shown in IFN-γR−/−
mice that had prolonged eosinophilia and Th2 cytokine production 35
. In this report, we show another role of IFN-γ in the regulation of Th2 responses. Th1 cell production of IFN-γ blocks Th2-induced inflammatory pathways downstream of cytokine production. This may occur by direct effects on eosinophils and epithelial cells or through an intermediate cell derived from the recipient mice. This mechanism of inhibition is of potential importance in asthmatic airways, where Th2 cells are chronically present, as we show that Th1 cells can inhibit Th2 cell effects while Th2 cells are actively secreting cytokines.
The inhibitory pathways induced by Th1 cells require more than a few days for induction. In our studies, the inhibitory effects of Th1 cells were seen in mice exposed to antigen over 9 d. These effects were not observed when we killed mice after just 2 d of antigen challenge: BAL eosinophilia was similar after transfer of Th2 cells or Th1 + Th2 cells and inhaled antigen (data not shown). It is possible that a short period of antigen exposure explains, in part, why other investigators did not observe a similar reduction in eosinophilia after recruitment and activation of Th1 and Th2 cells in the respiratory tract 3637
. This delay in inhibition of eosinophilia and mucus production by Th1 cells may point to a biological pathway that requires either time or stimulation with higher levels of IFN-γ to induce inhibition.
In defining the functional effects of Th1 cells on Th2-mediated airway inflammation, our studies provide insights into potential mechanisms governing symptom control in conventional allergy immunotherapy. These mechanisms need to be analyzed, as we have shown, by assessing the lymphocyte populations present at sites of inflammation where cytokines exert their effects. Studies of grass pollen immunotherapy for atopic skin disease and allergic rhinitis showed a reduction in late phase responses and eosinophil accumulation in the skin and nasal mucosa. These effects were associated with increased IFN-γ–expressing cells, yet cells positive for IL-4 and IL-5 were unchanged 910
. In addition, persistent Th2 cytokine production with increased IFN-γ–producing cells may explain why IgE levels and skin prick testing were not reduced, but IgG1/IgG4 levels increased in successful immunotherapy of pollen-allergic individuals 38
. Increasing the population of activated Th1 cells at sites of allergic inflammation should be a focus of new techniques of immunotherapy, as IFN-γ can exert its effects despite ongoing Th2 cell activation. These effects may be sustained, as there is evidence that potent, long-term stimulation with Th1 cytokines can shift a Th2-predominant population toward Th1 394041
Increasing Th1 cell activation during immunotherapy in asthma bears the potential risk of increasing inflammation. Successful immunotherapy for atopic skin disease and rhinitis in allergic patients has not borne out these concerns 1041
. In our studies and others, airway inflammation was not increased when both Th1 and Th2 cells were recruited to the lung in wild-type recipient mice 42
. This may relate to normal regulation of T cell proliferation that is typically present in intact, immunocompetent mice 43
. Hansen et al. 42
showed that Th1 and Th2 cells transferred into SCID mice, which lack these regulatory elements, leads to unrestrained proliferation and overwhelming airway and parenchymal inflammation. Furthermore, reducing airway eosinophilia leads to a reduction in tissue damage and cellular infiltration 444546
. Blocking recruitment of eosinophils to the airway results in a reduction in proinflammatory factors that potentiate inflammation. Thus, as these studies show, the net effect of recruiting Th1 cells to the lung is that total inflammation is relatively unchanged.
Airway inflammation has long been associated with excess mucus production. In chronic bronchitis, cystic fibrosis, and asthma, mucus hypersecretion is associated with different characteristic immune responses in the lung. Unlike asthmatics, patients with cystic fibrosis or chronic bronchitis typically do not show activated Th2 cells in their airways. In a model of asthma, we recently showed that Th2 cells stimulate airway mucus production 15
. Mucus induction requires IL-4Rα but is independent of IL-4, IL-5, eosinophils, and mast cells 47
. Other recent studies showed that IL-13 and IL-4 are important mediators in mucus production 4849505152
. Here we also show that Th1 cells stimulate mucus production in the absence of IFN-γR signaling. The neutrophil-predominant inflammatory response in these mice and the relationship of neutrophilia with mucus hypersecretion in cystic fibrosis and chronic bronchitis suggests that neutrophils may be involved in mucus hyperproduction. Neutrophil elastase is a potent mucus secretagogue 53
, yet the ability of this enzyme to stimulate increased mucin production has not been determined. We have shown that mucus induction is not due to a shift of the transferred Th1 cell population to a Th2 phenotype in IFN-γR−/−
recipient mice. There still remains the possibility that very low levels of IL-13 secreted by Th1 cells stimulate mucus production in IFN-γR−/−
mice. In summary, we have shown that mucus can be induced by an inflammatory response that is not dominated by production of IL-13 and IL-4.
Although different inflammatory responses stimulate mucus production, Th1 cells, through production of IFN-γ, inhibit mucus induced by both Th1 and Th2 cells. Inhibitory pathways for mucus have not been previously demonstrated. IFN-γ has been shown to have inhibitory effects on some epithelial functions. In gastric epithelial cells, mucus secretion was inhibited by IFN-γ 53
. IFN-γ also inhibited growth of a human bronchial epithelial cell line and reduced barrier function and chloride secretion in intestinal epithelial cells 5455
. Beyond these limited studies, the inhibitory effects of IFN-γ on airway epithelium are not known. Interestingly, mucus production is not a feature of Th1-mediated pulmonary diseases in humans. Mycobacterium tuberculosis
infection and sarcoidosis are diseases in which IFN-γ–producing CD4 T cells have been identified in the lung biopsies and in BAL 565758
. It is possible that the lack of mucus production in these conditions results from IFN-γ suppression. IFN-γ has many proinflammatory effects in the lung, most notably on macrophages, activating production of reactive nitrogen and oxygen species. The inhibitory effects of IFN-γ could therefore be through the production of inhibitory mediators by inflammatory cells or by direct effects on goblet cells. These studies establish the first known natural inhibitor of mucus production, one that can be active in different inflammatory settings.
In summary, using a transfer system that we developed to study the role of CD4 Th1 and Th2 cells in airway disease, we have defined two different pathways by which Th1 cells can regulate airway inflammation. Th1 cells, through an effect mediated by IFN-γR, block the recruitment of eosinophils to the airway and inhibit airway epithelial mucus production. In the absence of IFN-γR, Th1 cells induce mucus production but do not stimulate airway eosinophilia. Thus, Th1 cells have differential effects on stimulating these inflammatory responses. The inability of Th1 cells to stimulate eosinophilia likely results from a lack of IL-5. The mechanism by which mucus production is stimulated by Th1 cells in the absence of IFN-γR signaling is not yet known. As we learn how these inflammatory responses are regulated, we will identify new targets for directed immunotherapy for asthma and mucus hypersecretion.