HRF Transgenic Mice Phenotype Figure 11. Serum and BAL IgE Levels-Protocol III. Panel A illustrates the average total serum IgE +/2 SEM for each group of mice. Panel B is the average IgE levels detected in BAL supernatants from the same mice. IgE levels were measured by ELISA. N = 528 mice per group (backcrosses 6–8). doi:10.1371/journal.pone.0011077.g011 mechanism of action of HRF in our transgenic mouse model, and we are currently focused on the basophil. HRF has been previously shown to activate epithelial cells [18] and in our transgenic mouse model, HRF is expressed in the Clara cells of the epithelium. However, Clara cells do not produce IL-4, therefore, the observed increases in IL-4 are most likely due to secreted HRF. Also, as previously mentioned, HRF is known to be secreted in an ER/Golgi-independent manner [5]. Intracellular HRF levels have been associated with increases in tumors [38]. Elevations of intracellular HRF/TCTP that are associated with tumors would not likely occur in the three week time frame that we use in our model, and no gross changes in lung anatomy were observed. HRF transgenic mice have increased total serum IgE levels following either OVA sensitization or challenge (Figures 9 and 11), suggesting a systemic effect of the over expression of HRF in the lung. We also found increases in BAL IgE in transgenic HRF mice following antigen challenge (Protocol II) in the absence of sensitization, but not in the absence of antigen challenge (Protocol III), suggesting that HRF is active in the response phase. This is in accordance with the observation that OVA specific IgE in the serum is not increased in the HRF transgenic mice following OVA sensitization and challenge when compared to CC10 littermate controls treated the same way. Both observations suggest that HRF elicits its effects in the response and not the sensitization phase. References In summary, endogenous HRF is up-regulated after either allergen sensitization or challenge. Following OVA sensitization and OVA challenge, we see the same trends, namely, increases in IL-4 and eosinophils and no additional increases in AHR. However, there are no differences in total cells and increases in macropages following OVA sensitization and challenge with PBS. This points to the fact that HRF does not exert its effect in the sensitization phase alone. OVA challenge is necessary for HRF to exert a local effect in the response phase as measured by increases in BAL IgE levels. In contrast, either OVA sensitization or OVA challenge will suffice for HRF to have an effect on serum IgE levels. In general, HRF augments the inflammatory response to allergens. This has been seen in vitro in human studies over the past several years and is now seen in the mouse model of allergic inflammation. Future studies with this transgenic mouse model will elucidate the specific mechanisms of HRF’s actions on the inflammatory process with particular emphasis on the mouse basophil. Author Contributions Conceived and designed the experiments: JML SMM. Performed the experiments: YCY LX JML ACM SMM. Analyzed the data: YCY LX JML SYO ZZ SMM. Contributed reagents/materials/analysis tools: ACM. Wrote the paper: YCY LX JML SMM. 1. MacDonald SM, Lichtenstein LM, Proud D, Plaut M, Naclerio RM, et al. (1987) Studies of IgE-dependent histamine releasing factors: Heterogeneity of IgE. J Immunol 139: 506–512. 2. MacDonald SM, Rafnar T, Langdon J, Lichtenstein LM (1995) Molecular identification of an IgE-dependent histamine releasing factor. Science 269: 688–690. 3. Chitpatima ST, Makrides S, Bandyopadhyay R, Brawerman G (1988) Nucleotide sequence of a major messenger RNA for a 21 kilodalton polypeptide that is under translational control in mouse tumor cells. Nucleic Acids Res 16: 2350. 4. Yenofsky R, Cereghini S, Krowczynska A, Brawerman G (1983) Regulation of mRNA utilization in mouse erythroleukemia cells induced to differentiate by exposure to dimethyl sulfoxide. Mol Cell Biol 3: 1197–1203. 5. Amzallag N, Passer BJ, Allanic D, Segura E, The´ry C, et al. (2004) TSAP6 facilitates the secretion of translationally controlled tumor protein/histamine- releasing factor via a nonclassical pathway. J Biol Chem 279: 46104–46112. 6. Moldes M, Lasnier F, Gauthereau X, Klein C, Pairault J, et al. (2001) Tumor necrosis factor-alpha-induced adipose-related protein (TIARP), a cell-surface PLoS ONE | www.plosone.org 11 June 2010 | Volume 5 | Issue 6 | e11077