HRF Transgenic Mice Phenotype Figure 3. BAL Cell Counts for Mice in Protocol I. The average cell counts +/2 SEM from BAL supernatants for each group of mice is shown. 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. The insert shows mRNA for MCP-1 between various groups of mice. In all cases each group had between 4 and 11 mice (backcrosses 5–8). doi:10.1371/journal.pone.0011077.g003 as transgenic++ mice sensitized with PBS (++/PBS). The differences in histology between these groups are concordant with the differences in BAL cell counts. BAL cell assessments. BAL cells from transgene++ mice showed significant differences in the number of total cells as well as the number of eosinophils that infiltrated the lung (Figure 8 Panel A). Specifically, transgene ++ mice sensitized with PBS(++/PBS), and then challenged with OVA have more total cells than CC10 littermate controls treated the same way(CC10/PBS) (p = 0.04). No significant differences in specific cell types were seen between these 2 groups, although macrophages appear to be primarily responsible, as was found in antigen na¨ıve mice (Figure 3, Panel A). These data recapitulate the previous data from Protocol I indicating that HRF is capable of inducing the start of inflammation by recruiting cells to the lung. These data suggest that HRF operates independently of the effects of sensitization, and perhaps initiates the early steps of inflammation through macrophage recruitment. As expected, mice sensitized and challenged with OVA had increased eosinophils compared to littermate controls that received only PBS sensitization (p = 0.04) and the same was true for the transgene++ group comparisons (p = 0.005). Most importantly, the OVA sensitized and challenged transgenic++ mice (++/OVA) had a significantly larger eosinophil infiltration when compared to the CC10 litter mate controls treated the same way(CC10/OVA) (p = 0.02). These data support the previously published data that HRF is involved with the recruitment of eosinophils to the lung of allergen sensitized mice [29]. Figure 8 Panel B, pictures BAL cells from individual mice and illustrates the pattern seen for the groups as a whole shown in the graph in Figure 8 Panel A. IgE levels. Elevated IgE levels in the serum is a characteristic feature commonly seen in allergic asthma patients and in mouse models of allergen-induced asthma. IgE is important for the functions of cells bearing the high affinity IgE receptor, such as mast cells and basophils. As expected, we found significantly increased serum IgE levels following OVA sensitization and challenge in the transgene++ group and the CC10 group compared to littermate controls sensitized with PBS (p = 0.02 and p = 0.003 respectively, Figure 9, Panel A). Interestingly, transgene++ mice sham sensitized with PBS had significantly higher serum IgE levels than CC10 controls (p = 0.03). Similar to the cell count assessment (Figure 8, Panel A) HRF exerted an effect in the absence of allergen sensitization. There was no significant difference between CC10 and transgene ++ mice challenged and PLoS ONE | www.plosone.org 6 June 2010 | Volume 5 | Issue 6 | e11077