comparable significant differences were also detected when using the nonparametric Kruskal-Wallis test and Dunn’s post-test to compare the Cdyn responses generated in the control vs. OVA- sensitized rabbits in the absence (p,0.05) vs. presence (p = NS) of pretreatment with anti-Gbc blocking peptide. Gbc signaling mediates allergic airway inflammation in OVA-sensitized rabbits Together with its above bronchoprotective action, inhaled pretreatment with anti-Gbc blocking peptide also suppressed the pulmonary inflammatory response detected in the sensitized rabbits at 24 hr following OVA challenge. As shown by the representative lung sections in Fig. 3, relative to control (non- sensitized) rabbits that showed no sign of inflammation (Fig. 3A), lungs isolated from OVA-sensitized+challenged rabbits exhibited multiple patchy foci of peribronchial, perivascular, and parenchy- mal inflammation (Fig. 3B). The inflammatory cell infiltrates consisted mainly of neutrophils and, to a lesser extent, macro- phages and rare eosinophils, as depicted in the high magnification photomicrographs of representative lung tissue and BAL fluid (BALF) samples in Figs. 4A and 4B, respectively. In contrast to the lack of effect of pretreatment with MPS alone (Fig. 3C), the inflammatory response was distinctly suppressed to a similar extent in OVA-sensitized+challenged rabbits that were pretreated with either inhaled budesonide or anti-Gbc blocking peptide (Figs. 3D and 3E, respectively). Interestingly, by comparison, the inflamma- tory response was largely unaffected in OVA-sensitized+chal- lenged rabbits that were pretreated with gallein (Fig. 3F), suggesting differences between this small molecule inhibitor of Figure 2. Anti-Gbc blocking peptide prevents in vivo antigen- induced airway hyperresponsiveness in OVA-sensitized rab- bits. Relative to OVA-challenged control (non-sensitized; n = 4) rabbits, Rrs responses to MCh are significantly increased at 24 hr following antigen challenge in OVA-sensitized rabbits (n = 4). This heightened bronchoconstrictor responsiveness to MCh is suppressed in OVA- sensitized rabbits that are treated either with inhaled anti-Gbc peptide (1 mg/Kg; n- = 4) or budesonide (0.5 mg/Kg; n = 3) prior to antigen challenge. Data are mean 6 SE values. ANOVA used for multiple comparisons of mean Rrs values. *p,0.05; **p,0.01. Note: a significant difference is also detected when using the nonparametric Kruskal-Wallis test with Dunn’s post-test to compare the medians of the Rrs-max responses in the control vs. non-pretreated OVA sensitized rabbits (p,0.05), whereas no significant difference is detected between the control vs. OVA-sensitized animals that are pretreated with the anti-Gbc blocking peptide. doi:10.1371/journal.pone.0032078.g002 G Protein bc-Subunit Signaling in Asthma Gbc and the anti-Gbc blocking peptide with respect to their anti- inflammatory actions (see Discussion). Comparable results were obtained with respect to the corresponding changes in BALF cellular content and cytokine levels. Due to variability in recovery and dilution between the individual BALF samples, these data were analyzed with respect to changes in inflammatory cell counts per high power field (HPF) in BALF cytospins (Fig. 5A), wherein at least 500 cells were counted in each cytospin preparation, and as changes in the neutrophil/ macrophage cell ratio (Fig. 5B). The box plots in Fig. 5 depict the median and range of the values determined under each experimental condition, and the nonparametric Kruskal-Wallis test with Dunn’s post-test analysis demonstrated that, as compared to non-sensitized (control) rabbits challenged with OVA, the OVA-sensitized+challenged animals exhibited significant increases (p,0.05) in both the total number of cells (Fig. 5A) and neutrophil/macrophage cell ratio (Fig. 5B). Moreover, after antigen challenge, the control vs. OVA-sensitized rabbits also exhibited differences in their BALF levels of the signature Th1- and Th2-type cytokines, IFN-c and IL-13, respectively. Compared to controls, the levels of IFN-c were significantly reduced (p,0.05) in the OVA-sensitized+challenged rabbits (Fig. 5C), whereas the IL-13 levels (Fig. 5D) were significantly increased (p,0.05). As further depicted, the induced changes in the above inflammatory indices were suppressed in OVA-sensitized+challenged rabbits that were pretreated with inhaled anti-Gbc blocking peptide, with no sigificant differences detected when compared to the control animals. Gbc activation regulates PDE activity in OVA-challenged allergic lungs and sensitized ASM tissues Upregulated PDE4 activity was shown to play a determinant role in mediating airway hyperresponsiveness and inflammation in response to allergen challenge in asthmatic individuals [28] and in animal models of allergic asthma [29–33]. Given this evidence, together with the above data implicating an association between Gbc activation and rolipram-sensitive proasthmatic changes in responsiveness in OVA serum-sensitized ASM tissues (Fig. 1), we next examined whether lung tissues isolated from OVA-sensiti- zed+challenged rabbits and ASM tissues passively sensitized with sera from these animals exhibit Gbc-dependent changes in cAMP PDE activity. Relative to peripheral lung tissue sections isolated from control rabbits, as well as sensitized rabbits that were not challenged with OVA, significantly increased levels of PDE activity were detected in the lung tissues of OVA-sensitized+chal- lenged rabbits (Fig. 6A). Similarly, relative to na¨ıve ASM tissues exposed to control serum, significantly increased levels of PDE activity were detected in ASM tissues that were passively sensitized with OVA serum (Fig. 6B). This upregulated PDE activity was abrogated in lungs from OVA-sensitized+challenged rabbits that were pretreated in vivo with anti-Gbc blocking peptide, and the latter pretreatment also suppressed the increased PDE activity in OVA serum-exposed ASM tissues (Figs. 6A and 6B, respectively). Of note, comparable results were also obtained in separate experiments wherein na¨ıve ASM tissues were exposed for 24 hr to a maximally effective concentration of IL-13 (50 ng/ml) [34], the Th2 cytokine that is critically implicated in mediating antigen- induced airway hyperresponsiveness in vivo [35,36], as well as the pro-asthmatic changes in contractility in atopic serum-sensitized ASM tissues, the latter attributed to IgE-induced activation of its low affinity receptor (CD23) [24,34]. As shown in Fig. 6C, relative to controls, IL-13-treated ASM tissues exhibited significantly increased PDE activity that was suppressed by pretreatment with either the anti-Gbc blocking peptide or gallein (10 mM). PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e32078