HRF Transgenic Mice Phenotype Figure 8. BAL Cell Counts. Panel A depicts the average cell counts +/2 SEM from BAL supernatants for each group of mice. Total cells were counted using Erythrosin B stained under light microscopy. Cell differentials were done using cytospin slides of the BAL stained with Dif-Quik. Panel B shows representative cytospins from these mice with genotype and sensitization reagent on the top of the cytospin. N = 629 mice per group (backcrosses 3–6). doi:10.1371/journal.pone.0011077.g008 macrophages have been shown to secrete HRF in response to M- CSF [29]. Therefore, activated macrophages could be a source of endogenous HRF found in the lung. In addition, OVA immunized mice challenged with M-CSF caused macrophage infiltration and HRF over production that was similar to OVA challenge. [29]. Furthermore, HRF has been shown to stimulate the secretion of GM-CSF and IL-8 from bronchial epithelial cells [18]. Taken together our data and the previously mentioned studies suggest that HRF may increase airway inflammation by activating cells such as epithelial cells or basophils (see below) to increase cytokine and chemokine production which in turn up regulate the HRF secretion and significantly contribute to the local inflammation in the allergic, asthmatic lung. We found that HRF levels in the lungs were increased by both OVA sensitization and challenge. This increase was observed in both HRF transgenic mice and CC10 control mice following either OVA sensitization or OVA challenge (see Figure 4). The immunoflourescent staining of the mouse lung (Figure 5) illustrates both an increase in density and intensity of staining suggesting both an increase in the number of cells expressing HRF and the amount of HRF expressed. The increase in expression in transgenic mice could be due to the increase in the number of Clara cells following OVA challenge and sensitization. However, GFP expression levels, as analyzed at protein and mRNA levels, were not increased under these same conditions. Moreover, the increases following OVA sensitization or challenge in the CC10 littermate controls could be due only to endogenous HRF. We hypothesize based on this data as well as the previously mentioned work, that antigen challenge could increase levels of HRF which then acts on other cells to produce inflammatory cytokines. We also showed after OVA sensitization and challenge that HRF transgenic mice had increased levels of the TH2 cytokine IL4, but not eotaxin, IL-5 or IL-13. IL-4 is necessary for the induction of IgE production and the increases may be responsible for the observed increase in total IgE. In addition, IL-4 has been shown to play an important role in the migration of eosinophils from the lung into the airway [27,28]. Moreover, previous studies using human cells have shown that HRF is capable of inducing IL- PLoS ONE | www.plosone.org 9 June 2010 | Volume 5 | Issue 6 | e11077