We have established that highly purified human blood eosinophils express IL-7Rα (CD127) and that IL-7 stimulation of eosinophils leads to phosphorylation of STAT5. Moreover, we demonstrated a novel function of IL-7 for enhancement of human eosinophil survival and upregulation of the activation marker CD69. An in vivo relevance for IL-7 was suggested by the observation that levels of IL-7 were highly correlated with eosinophils in BAL fluid of allergic asthma subjects 48 h after airway allergen challenge. These data suggest that IL-7 may play a role in allergen-induced eosinophilic airway inflammation that is associated with asthma.
The primary function of IL-7 is considered to be regulation of the development and survival of T cells; however, there have been sporadic reports that IL-7 can also affect nonlymphoid cells. For example, IL-7 has been shown to activate human monocyte tumoricidal activity and to induce their production of IL-6, IL-1, TNF-α and IL-8 (13
). Although a direct effect of IL-7 on mature eosinophils has not been previously reported, IL-7 has been shown to stimulate eosinophil colony formation from bone marrow cells (16
). There is also indirect in vivo
evidence associating eosinophils and IL-7. Eosinophils have long been noted in the thymus (38
), which is a principal location for IL-7 production. Although there is growing evidence that eosinophils can influence T cell function (19
), the significance of eosinophils at this T cell site remains a mystery. Eosinophil infiltrates have been reported in murine tumors engineered to overexpress IL-7 (40
) and in colonic mucosa of mice with selective over-expression of IL-7 in the colon (42
). Conversely, eosinophils were lacking in a mouse model of colitis with targeted IL-7 deletion (43
). Despite these scattered inferences to eosinophils and IL-7, to our knowledge, this is the first report to demonstrate the IL-7Rα chain on eosinophils and to show their activation by IL-7.
Evidence that IL-7 can activate eosinophils included induction of STAT5 phosphorylation, stimulation of morphological changes consistent with cellular activation/migration, upregulation of CD69 on the cell surface, and enhanced eosinophil survival. The effects of IL-7 were significantly reduced in the presence of blocking antibodies to GM-CSF, but not anti-IL-5 suggesting that IL-7 activation of these particular eosinophil functions is mediated via autocrine production of GM-CSF. Neutralization of GM-CSF reduced STAT5 phosphorylation within 10 min of eosinophil exposure to IL-7. The rapid kinetics of the anti-GM-CSF-mediated effect raises the possibility that IL-7 can induce autocrine release of preformed GM-CSF. Indeed, there are reports that GM-CSF is stored in crystalloid granules of eosinophils (44
). The kinetics of inhibition of GM-CSF activity is reminiscent of eosinophil storage and rapid release of preformed RANTES (45
) and IL-4 (46
), which are reported to be stored in specialized small secretory vesicles and selectively released by a process known as piecemeal degranulation (47
). Bandeira-Melo and colleagues showed that eotaxin-induced IL-4 release could not be measured by ELISA, but was instead detected by a unique gel-based dual antibody detection assay (49
). Thus, the fact that we were not able to directly detect GM-CSF production by eosinophils is not unexpected. A number of reports have demonstrated that eosinophil survival induced by factors such as IL-15 (33
), hyaluronic acid (32
), or TNF-α plus fibronectin (32
) can be reversed by neutralization of GM-CSF, yet demonstrable levels of GM-CSF could not be measure by ELISA. Furthermore, it has been shown that the continuous presence of minute amounts of GM-CSF are sufficient to inhibit eosinophil apoptosis (30
We recognize that there are inherent limitations to our studies. First, nanomolar concentrations of IL-7 were necessary for activation of eosinophils. This observation is consistent with what has been reported for IL-7-induced release of GM-CSF by T cells (50
). The reason why GM-CSF release by T cells and eosinophils requires relatively high concentrations of IL-7 is not known. There is evidence that human T cells express both high and low affinity IL-7 receptors (51
). It is possible that IL-7-induced GM-CSF release occurs through activation of low affinity receptors, and thus requires higher concentrations of IL-7 to achieve effective cell signaling and functional cellular responses. The report that IL-7 can stimulate GM-CSF production by T cells (50
) raises a second potential limitation. It is possible that the small numbers of T cells or other mononuclear cells present in our eosinophils preparations (generally <2%) contribute to the effects of IL-7 on eosinophils. This scenario is unlikely based on our observation that an eosinophil preparation of 99.9% purity responded to IL-7 and addition of 3% PBMC did not further enhance IL-7-induced eosinophil survival, expression of CD69 or levels of STAT5 phosphorylation. Finally, although we have clearly demonstrated the presence of CD127 mRNA expression (17
) and the protein by Western blot analysis (), the detectable level of the receptor by flow cytometric analysis is low and inconsistent. The fact IL-7Rα is detected by immunoblotting at levels between 26 and 84% of the expression in an equal mass of a standard lymphocyte preparation and that eosinophils respond to stimulation with IL-7 with respect to multiple biological readouts, is consistent with an IL-7 receptor conformation in eosinophils that limits binding or accessibility of the monoclonal antibodies used for flow cytometry. We suspect that in eosinophils, the receptors may be sequestered in multi-protein complexes, caveolae or in other cellular microdomains that mask the epitope detected by the monoclonal antibody used for flow cytometric analysis.
In concert with our in vitro
studies demonstrating a direct effect of IL-7 on eosinophils, we have also shown that airway eosinophilia induced by allergen challenge of allergic asthma subjects is associated with increased BAL fluid concentrations of IL-7. The source of IL-7 in BAL fluid was not determined in this study. Epithelial cells are the principal source of IL-7 in tissues where IL-7 is predominant including the thymus, bone marrow, and intestine (12
). While there is a paucity of studies on IL-7 in the airway, there is some evidence that BAL cells express IL-7. An early report documented IL-7 mRNA in BAL cells from lung transplant patients (52
) and a more recent study demonstrated IL-7 staining of alveolar macrophages and epithelial cells in cytospin preparations of BAL cells from patients with concurrent infection with HIV-associated tuberculosis (53
). Further studies of BAL cells and bronchial biopsies are required to determine the source of IL-7 in the airway of asthma subjects following allergen challenge. There is also a need to determine if these cells are directly activated by allergen or indirectly affected by factors produced by other IL-7-responsive cells such as T cells and monocytes. For example, IL-7 synthesis by epithelial cells can be upregulated by IFN-γ, IL-1, or TNF-α (54
The presence of IL-7 adds to the redundancy of eosinophil survival factors in the airway after allergen exposure. In asthma, one could envision that IL-7 may provide a pathway for GM-CSF production and enhanced survival of eosinophils in the event of deficient production by prominent GM-CSF-producing cells such as airway epithelial cells and/or alveolar macrophages. Furthermore, the ability of IL-7 to promote eosinophil survival and induce the eosinophil survival factor GM-CSF may contribute to eosinophilia in nonatopic airway diseases that are not typically associated with increased levels of the eosinophil-specific cytokine IL-5.
In summary, we have established a novel function for IL-7 in the activation of eosinophils and potentiation of their survival, and have demonstrated that levels of IL-7 in BAL fluid are increased by airway allergen challenge and parallel the influx of eosinophils, thus supporting the notion that IL-7 contributes to airway inflammation by promoting eosinophil activation and survival.