The contribution of IL-4, IL-13 and IL-4Rα to allergic asthma disease progression and pathological characteristics has been examined in some detail [7
]. However, the role of AAM in allergic lung inflammation has remained unclear. Several studies, including our own, have demonstrated a correlation between the presence of IL-4Rα+ macrophages and enhanced eosinophilic inflammation using infection and allergic models in mouse and man [16
]. Previous studies have used clodronate loaded liposomes to deplete macrophages to demonstrate their role in infection-induced inflammation [35
]. While informative, this approach is limited because clodronate can deplete any phagocytic cell, not only macrophages; the liposomes can induce a confounding inflammatory response on their own; and the depletion only lasts for 3 days [54
In this study, we tested whether IL-4Rα+ macrophages were sufficient to enhance allergen-induced eosinophilic inflammation. Since we provided OVA-specific, primed TH2-effectors, additional exposures to OVA would mimic allergen-induced exacerbation. Using this approach, we found that adoptive transfer of IL-4Rα+/+ BMM, but not IL-4Rα-/- BMM, significantly enhanced the TH2-dependent, OVA-induced recruitment of eosinophils to the lung. As would be expected in this model of inflammation, we found little to no inflammation in the absence of TH2 cells. Thus, in the presence of TH2 cells, transfer of IL-4Rα+ macrophages is sufficient to enhance inflammation. However, it is not entirely clear whether they are the primary effectors of the enhanced inflammation or whether they stimulate another cell type.
We found that lungs from OVA-challenged IL-4Rα-deficient mice that received TH2 cells and IL-4Rα+ BMM showed positive staining for YM1/2. Immunohistochemistry of serial sections and immunofluorescence showed substantial overlap in staining of macrophages with YM1/2 and Mac3. The F4/80 staining showed a broader profile, overlapping with the YM1/2 and Mac3 staining, but also staining other inflammatory cells, especially cells cuffing blood vessels. Our results are consistent with those from the Nb
infection model showing that Mac3+ cells express AAM markers [58
]. However, whether Mac3 will be a reliable cell surface marker for AAM in inflamed tissues remains to be firmly established. Using immunofluorescence microscopy, we observed an interesting heterogeneity in expression of Mac3 and YM1/2 with Mac3-
, and Mac3+
cells present in the inflamed lungs. Whether these different subsets mediate different effector functions is unknown.
AAM have been shown to participate in other TH2 mediated inflammatory responses. Mice infected with nematode parasites induced the localized development of AAM in response to IL-4 [32
]. Recruitment of eosinophils to the lung and peritoneal cavity in mice infected with Nb
was dependent on macrophages; the absence of macrophages greatly prevented eosinophil recruitment [34
]. Furthermore, intratracheal administration of in vitro differentiated AAM elevated the typically low level of allergic inflammation seen in male mice [39
]. Taken together, our results and those from the other parasite infection models and allergic lung inflammation models strongly suggest that AAM are important, active contributors to inflammation and are not just bystander cells responding to the TH2 cytokines.
In our BMM transfer model, all host cells were IL-4Rα-/-
, including resident lung macrophages, and thus were unable to respond to IL-4 or IL-13. We detected IL-4Rα+ macrophages in the lungs of mice that received IL-4Rα+/+
BMM by intraperitoneal injection. In addition, we detected YM1/2+ macrophages in the lungs of these mice, but not in lungs of mice that received BMM lacking the IL-4Rα. These results show that the transferred BMM likely respond to the IL-4/IL-13 produced by the TH2 effectors in vivo and can localize to the lung. This is in contrast to studies using a parasite infection model reported by Jenkins et al. [59
] where the majority of lung AAM was derived from proliferating resident lung cells and not from new immigrants. It is possible resident tissue macrophages proliferated in our model, however, since they lacked IL-4Rα, they would not be able to differentiate into AAM. The relative contribution of AAM derived from newly arrived inflammatory macrophages versus AAM derived from resident lung macrophages to eosinophilic inflammation remains to be established.
An important question arising from these studies is how the IL-4Rα+
macrophages actively enhance the inflammatory response. IL-4/IL-13 stimulated macrophages make a number of factors including high levels of arginase 1, and chitinase and chitinase-like family members, however their contribution to allergic inflammation is not well understood [17
]. Deletion of arginase 1 had no effect on the level of allergic inflammation [60
]. Deletion of the chitinase family member brp39 profoundly suppressed allergic inflammation by an unknown mechanism [61
]. Another chitinase like family member, YM1/2 has been shown to enhance TH2 cytokine production by T-cells [62
IL-4/IL-13 stimulation of macrophages induces the production of eotaxin 1 and 2 (CCL11 and CCL24) that are important for the chemotaxis of eosinophils [29
]. This chemokine production is amplified by IL-33 leading to enhanced eosinophilic inflammation [37
]. AAM also produce MDC (macrophage-derived chemokine; CCL22) and TARC (thymus and activation-regulated chemokine; CCL17) that recruit macrophages and TH2 cells respectively [20
]. IL-4 stimulation of AAM has been shown to enhance the production of MIP-1α (macrophage inflammatory protein-1; CCL3) a granulocyte chemokine [29
]. The ability of AAM to produce chemokines may explain the enhanced recruitment of eosinophils we observed in mice receiving IL-4Rα+/+
macrophages. We observed increased amounts of eotaxin-1, RANTES, and MCP-1 in the BAL fluid of mice receiving IL-4Rα+/+
BMM, although the precise cellular source of these chemokines is not known. They could be derived from the AAM present in the lung and the BAL or they could be derived from another cell type responding to products made by the AAM.
In this regard, Medoff et al. found that an F4/80-
myeloid cell type was able to produce chemokines in a STAT6-dependent manner that enhanced both
T-cell and eosinophil recruitment to the lung [63
]. Since AAM have been shown to produce IL-13, it is possible the AAM are indirectly responsible for the increase in chemokines found in the BAL by producing IL-13 that can act on other myeloid cell populations in the lung [35
]. Another group has shown that AAM regulate the activity of myeloid dendritic cells in female mice, suggesting a mechanism whereby females are more susceptible to lung inflammation than males [39
]. While not a focus of this study, it is possible products made by AAM enhance the antigen presenting function of dendritic cells leading to enhanced T-cell activation. Clarification of the specific myeloid/macrophage population directly responsible for the enhanced eosinophilic inflammation observed after IL-4Rα+
BMM transfer will require further investigation.
AAM may provide an environment hospitable to TH2 cells establishing a reciprocal relationship between these two cell types during the course of allergic inflammation. Such a relationship has been described as the innate-adaptive axis in asthma [35
]. Chemokines produced by AAM such as TARC (CCL17) may enhance the recruitment of TH2 cells to the site of inflammation. Additionally, AAM increase expression of the IL-1 receptor antagonist (IL-1Ra), a decoy for IL-1 [17
]. AAM can also produce IL-10 [17
]. IL-10 suppresses IFNγ and STAT1 regulated responses. Since IFNγ can suppress TH2 differentiation, the IL-10 may act to perpetuate the Type 2 inflammatory response. Production of IL-1Ra and IL-13 by AAM may suppress the differentiation and function of TH17 cells [64
]. In our model, where we provide high numbers of in vitro differentiated TH2 cells exogenously, these regulatory processes may not be apparent. We did not see a difference in the number of T-cells in the lungs or a difference in the amount of IL-4 or IL-5 in the BAL fluid between the IL-4Rα+ or IL-4Rα- transfer groups suggesting that the TH2 cells were being recruited and activated sufficiently in both settings. Additionally, there was no difference in the amount of IFNγ in the BAL fluid, while the levels of IL-17 were below detection in all groups (data not shown).
However, we did observe a significant increase in the levels of TNFα in the BAL of mice receiving IL-4Rα + BMM. While high levels of TNFα are typically associated with TH1-and TH17-mediated inflammatory diseases, TNFα levels have also been correlated with allergic inflammation in mice and with asthma severity in humans (reviewed in [65
]. TNFα could play a role in the pathogenesis of asthma by stimulating a number of responses in inflammatory cells and structural cells including the recruitment and activation of eosinophils, the activation of mast cells, and the upregulation of adhesion molecules on endothelial and epithelial cells. The cellular source of our observed increase in TNFα in the BAL is unknown. Similar to the chemokines, many cell types are capable of producing TNFα including T-cells, macrophages, eosinophils, and epithelial cells [65
]. AAM have also been shown to produce TNFα which can be enhanced by bacterial infection or LPS treatment [66
]. Thus, it is possible the full set of cytokines and chemokines produced by AAM could favor TH2 dominance and promote a robust inflammatory environment in the lung.