In the past decades, tremendous progress has been made in the understanding of the effector phase of allergic airway diseases. However, the sensitization phase of these pathologies is far less understood, partly due to the lack of suitable animal models (1
). In recent years our laboratory has contributed to the understanding of the priming phase of allergic airway disease by developing different models suited to analyze this part of the immune response in which we show 1) a requirement for TLR and MyD88 signaling in priming via the airways (3
), and 2) a promotion of naive T cell priming through an ongoing Th2-polarized airway inflammation (9
), findings that have since been confirmed by other investigators (5
Combining these two major findings, we show herein that IL-4 can bypass the need for signals via the immune system in pulmonary priming, a process we call IL-4-dependent pulmonary priming. Similar to what has been described for other cytokines, for example, TNF-α (3
) or GM-CSF (19
), IL-4 induces CD4+
T cell priming toward new Ags. Through consecutive studies, we were able to determine that this process depends on intact expression of IL-4Rα on CD4+
T cells and myeloid DCs but also on yet undefined cells in the structural compartment of the lung or mLN. While the identification of the cell types and mechanisms involved in IL-4-dependent pulmonary priming in the structural compartment was beyond the scope of this study, our results suggest that IL-4 contributes to Th2 priming via the hematopoietic compartment by 1) an autocrine pathway directed toward homing of naive T cell to local LNs, as suggested by other studies (10
), and 2) by activating lung DC and facilitating their interaction with T cells in the draining LNs, a finding that had also been suggested by older studies (28
It is well known that IL-4 and the IL-4 receptor play a central role in the allergic airway response and Th2 differentiation in general (11
), crucially regulating the occurrence and intensity of airway inflammation by expression of the IL-4 receptor on hematopoietic and nonhematopoietic cells (32
). Until recently, however, it was not clear if IL-4 can directly influence naive T cell priming in vivo. In vitro studies had shown that IL-2 is necessary for the initial clonal expansion during priming and/or differentiation of Th2 cells, and its autocrine secretion was thought to be crucially dependent on two-signal-dependent T cell activation by fully activated APCs (33
). Activation of APCs, on the other hand, was thought to depend on the recognition of stimuli directed toward the innate immune system (3
). Previously, only our collateral priming system (9
) demonstrated that IL-4 can facilitate T cell priming toward new Ags independent of signals via the innate immune system.
Our new data assert that priming via the airways can occur independently of signals via pattern recognition receptors. Other authors have determined that priming within the airways is driven by pathogen-associated molecular patterns recognized by TLR2 (6
), TLR3 (38
), and TLR4 (3
). However, our new results confirm and extend the data from Eisenbarth et al. (9
) and Hayashi et al. (10
) that priming at this site can also occur independently from such signals and therefore establish IL-4-dependent pulmonary priming as a novel mechanism of sensitization via the airway.
Furthermore, by showing a direct effect of IL-4 on naive T cells and via DCs in naive mice, we establish the main cellular players in this process. Previous studies have shown that the facilitation of priming toward new Ags through an ongoing airway inflammation crucially depends on T cell recruitment to the draining LN (10
), an observation we retrace with our findings that recruitment of naive T cells to the mLN is affected by IL-4 but not proliferation per se (). By showing that within the hematopoietic compartment IL-4Rα expression on CD4+
T cells alone is sufficient to induce secretion of Th2 cytokines and low-grade airway inflammation (), two aspects of the asthmatic phenotype, but that is is insufficient to fully induce these two features (e.g., systemic sensitization and airway inflammation), whereas IL-4Rα expression on DCs alone is insufficient to induce any of the hallmark phenotypical changes of our asthma model, we also recapitulate older data showing that CD4+
T cells are the single hematopoietic cell type sufficient for development of the main features of the asthmatic phenotype and crucially depend on IL-4 in this context (15
). Considering that more recent studies show that IL-4 renders naive T cells resistant to T regulatory-dependent suppression (39
) and down-regulates IDO expression (40
), it is tempting to speculate that the modulation of regulatory mechanisms contributes to IL-4-dependent pulmonary priming.
Moreover, various studies have shown that airway inflammation and IL-4 can activate immature DCs in vivo and in vitro (29
). Data on the contribution of the IL-4 receptor has indicated that cells with a phenotype also compatible with immature DCs play a crucial role in regulating the severity of airway inflammation (32
), a finding compatible with our observations. Our transfer experiments () suggest that DCs capable of responding to IL-4 are necessary to induce the maximal asthmatic phenotype but are insufficient to induce any of the phenotypical changes of our model by themselves, contrary to our observations when transferring T cells alone (). Furthermore, our results suggest that increased DC recruitment to the lung is mediated, at least partially, to an increase in CCL20 secretion, which is known to be secreted by epithelial cells in response to IL-4 (42
) and that has been shown to play an important role in DC migration (43
). The dissection of these mediators with particular emphasis on the contribution of the structural, nonhematopoietic compartment and the recruitment of T cells and DCs to the draining LNs is the topic of ongoing studies in our laboratory.
Our results demonstrating that proliferation of Ag-specific transgenic T cells was not affected (, ) despite increased Ag-presenting capacities of DCs within the draining LNs () are difficult to reconcile. Our present data are not sufficient to explain these discrepancies. However, our data showing that increased numbers of CD4+ and CD8+ T cells are present in the lymph nodes of animals treated with OVA and IL-4 evoke the possibility that the small number of transgenic T cells is not so much affected by a pro-proliferative property of IL-4 as the proliferation of endogenous T cells. More detailed experiments are needed to address this issue.
In summary, our studies extend the known findings pertaining to DCs and IL-4 by suggesting that through increased recruitment, maturation, and Ag presentation IL-4 might influence DC-dependent priming of naive T cells for new Ags. Th2 differentiation by DCs has been shown to be crucially dependent on NF-κB induction in DCs (44
), which in turn can be induced by maturation with IL-4 (45
), suggesting a molecular mechanism for our observations, which await analysis in future studies.
In vitro and in vivo studies have shown disparate results with regard to the effects of IL-4 on the capacity of DCs to influence the balance of Th1 or Th2 polarization (44
). They reveal a complex regulatory role of IL-4 depending on the timing, culture conditions, mediators, and cell types studied. In our system, similar to that of Sriram et al. (49
), the data point toward a positive role for IL-4 in driving an immune response toward Th2 polarization. Our in vivo data showed an increase in Th2 cytokines () as an endpoint read-out parameter influenced by the capacity of DCs to prime T cells toward Th1 or Th2 polarization. Similarly, in vitro data confirmed an increased capacity of DCs subjected to priming with IL-4 in vivo to prime naive T cells (data not shown). However, future studies will have to determine molecular pathways and mediators utilized by these DCs to induce the phenomena that we observed.
The clinical implications of our findings relate to how susceptible individuals might acquire new sensitizations to environmental proteins. Although the interaction between genetic susceptibility and environmental factors during the development of allergic diseases in childhood is recognized to play a pivotal role in the development of these diseases (50
), this concept places little emphasis on the contribution of the local microenvironment. Our data suggest that this environment might provide key factors to the future direction of the disease once a susceptible individual has become sensitized. Furthermore, it might also determine an individual's susceptibility to develop an allergic response in the first place. Clinical data show that sensitization to one Ag is a major risk factor for subsequent sensitizations to other Ags (51
), a process that is associated with an increase in IL-4 production from PBMC (54
). We suggest that the initiation of Th2-polarized airway disease and the subsequent clinical outcome of an individual crucially depend on the genetic propensity for IL-4 production as well as environmental factors triggering IL-4 production in the organ where allergen contact occurs. Our model clearly shows that IL-4 facilitates priming of CD4+
T cells for new Ags, suggesting that interventions aimed at reducing IL-4 production such as immunotherapy (55
) might take up the pathological immune response at a crucial point and would therefore be effective in the long-term inhibition of disease progression.