In this report, we investigated the role of neutrophils in the innate immune response to allergen, specifically related to the role of allergen-derived proteases and PAR-2. Our results suggest that allergen exposure upregulated PAR-2 on pulmonary neutrophils and that PAR-2 activation resulted in neutrophil-derived TNFα release. We have previously reported that a very early innate immune response occurred following allergen exposure. Within one hour, a significant release of TNFα in the BAL fluid is detected  and while this is maximal at 6 h post exposure, levels remain significantly higher than PBS controls up to 24 h later . Neutrophil-derived TNFα production is important as we have shown that depletion of neutrophils prior to allergen exposure abolished GC frass-induced TNFα production when assessed 18 h later . To our knowledge, this is the first study to address the role of cytokine production from PAR-2-deficient pulmonary neutrophils. Expression of PAR-2 was important for maximal expression of TNFα from pulmonary neutrophils. While we did not directly study this, it is likely that PAR-2-mediated TNFα production from neutrophils is mediated by nuclear factor (NF)-κB and extracellular regulated kinase (ERK). We have recently demonstrated that activation of PAR-2 regulated TNFα production by NF-κB and ERK, but not p38, in alveolar macrophages . Other reports have also shown that PAR-2 activation leads to increased ERK and IκBα/NF-κB signal transduction pathway activation [14–16]. In our study, we report that the major consequence of PAR-2 activation on neutrophils is the release of TNFα in the airways.
Vergnolle et. al. showed that PAR-2 contributed to the early events of inflammation by playing a crucial role in leukocyte recruitment and extravasation . In that study, selective activation of PAR-2 significantly increased leukocyte rolling and leukocyte adhesion to the endothelium. A subsequent study showed that leukocyte rolling was significantly lower in PAR-2-deficient mice compared to wild type mice in a model of acute inflammation . In this report, we found that PAR-2-deficient mice were less responsive to GC frass in their ability to recruit neutrophils into the lungs and BAL fluid; however while this was statistically significant, the levels of neutrophils in the PAR-2-deficient mice compared to wild type were not completely repressed. While it is currently unclear from the previous studies [17, 18] whether PAR-2 expression was crucial on the leukocyte or the endothelium, it is clear that PAR-2-deficient mice have a somewhat altered ability of neutrophil recruitment into the lungs and airways following allergen exposure. It is also important to note that in the PAR-2-deficient mice, we have previously reported a decrease in the neutrophil chemoattractant KC in the BAL fluid of mice following allergen exposure , which could also play an important role in neutrophil recruitment into the lung. These data are similar to those presented by Williams et al.  who found that KC levels in the BALF of PAR-2 mice were significantly lower following LPS than in wild type mice. Interestingly we found that MIP-2 release was unaltered in the PAR-2-deficient mice 18 h post allergen exposure. A recent report found an early and transient regulation of MIP-2, where MIP-2 release was reached a peak at 3 hr post inhalation and reached basal levels by 12 hr. In that study, the presence of PAR-2 regulated MIP-2 expression only at 3 hr post LPS exposure in the lung homogenate but not in the BALF . It is currently unclear of the role MIP-2 would play in mediating neutrophil recruitment into the airways. Our current study cannot clearly identify if PAR-2 plays a role in extravasation of the neutrophil or the chemoattraction of neutrophils into the lung and thus further studies are required to answer this question.
Recently it was shown that neutrophils may act as professional antigen-presenting cells (APC) by inducing their expression of MHC class II and co-stimulatory molecules CD80 and CD86 and by processing and presenting antigen to trigger T-cell activation . Based on that study, we investigated the levels of MHC class II, CD80 and CD86 on pulmonary neutrophils following a single exposure to GC frass and found that the lack of PAR-2 had no effect on the expression of these molecules. We have recently reported that on pulmonary mDCs, the expression of CD80 and CD86 was somewhat dependent on the presence of a functional PAR-2 . In PAR-2-deficient mDCs, the expression of these co-stimulatory molecules were slightly, but significantly decreased. PAR-2 activation has been shown to enhance the maturation of bone marrow-derived DCs  suggesting the potential role for PAR-2 activation on the development of DCs as APC. It is still not completely clear what the overall relevance of neutrophils as APC has on the initiation of allergic airway inflammation, and further studies are needed in this area. However, in this report, we find that PAR-2 expression did not appear to be important for allergen uptake or phagocytosis of the allergen, nor did it appear to play a role in co-stimulatory molecule regulation on the neutrophil.
Many cells are likely involved in the initiation of the innate immune response, including the neutrophil, which we have consistently seen in high numbers following allergen exposure [7, 10, 13]. It is still unclear what the overall role of the neutrophil is in the initiation of allergic airway inflammation. In a guinea pig model of OVA-induced asthma, removal of neutrophils was found to decrease mucus production by preventing goblet cell degranulation . Neutrophils also release interleukin (IL)-8, growth-related oncogene α (GRO-α), macrophage inflammatory protein 1-α (MIP-1α) and MIP-1β . IL-8 and GRO-α act to recruit additional neutrophils, while MIP-1α and MIP-1β are chemoattactive for immature DCs and T cells. Thus, the neutrophil can play a direct role in altering the cellular milieu following stimulation.
We found that pulmonary neutrophils release a substantial amount of TNFα following allergen exposure. We showed this in two ways, first by isolating neutrophils from whole lung and treating them ex vivo with allergen, and second by directly isolating pulmonary neutrophils from BAL fluid following allergen exposure. The second method is more physiologically relevant in that we identified the amount of TNFα release from airway-derived neutrophils stimulated with GC frass in vivo. For this experiment, we counted the cells and cultured them in equal quantities, so it is likely that since there are less neutrophils in the airways of PAR-2 mice, the overall amount of neutrophil-derived TNFα in the airways will be even less. In a previous study, we found higher levels of TNFα in the BAL fluid of mice following allergen exposure so it is likely that other lung cells are also involved in TNFα release. Recently, TNFα was shown to enhance TGF-β1-driven epithelial-to-mesenchymal transition suggesting that TNFα could play a crucial role in the reprogramming of epithelial cell responses . In patients with asthma, increased TNFα levels have been detected in the airways , and there is some evidence that increased airway TNFα may play a role in refractory asthma . It is unclear what the major cellular source of this TNFα is, however there are a subset of asthma patients with refractory asthma that have significantly increased levels of neutrophils . What is still unclear is the direct role of neutrophil-derived TNFα in modulating allergic airway inflammation.
The importance of PAR-2 in modulating allergic airway inflammation has recently been shown. PAR-2 mice exhibit decreased airway hyperresponsiveness, serum IgE and Th2 cytokine production following allergen sensitization and challenge compared to wild type controls [4, 28]. Our collective data has shown an important role for GC frass-associated proteases and PAR-2 in modulating cytokine production from alveolar macrophages , airway epithelium , myeloid dendritic cells , and in the current study, neutrophils; all of which are important activators of the innate immune response. To our knowledge, this is the first report investigating the role of PAR-2 in mediating the activation of airway neutrophils following exposure to an allergen.