Figure 3
From: Activation of C3a receptor is required in cigarette smoke-mediated emphysema
Expression of C3aR in mouse lung inflammatory cells and human mDCs. WT and C3-deficient mice were exposed to cigarette smoke or air for 6 months. (a) Expression of C3ar1 mRNA in BAL cells isolated from WT or C3−/− mice exposed to air or cigarette smoke was measured by qPCR. ***P<0.001 as determined by the one-way ANOVA with Bonferroni’s multiple comparison. Representative (b) and cumulative data (c) measuring C3aR MFI in single lung cells gated on B220− CD11c+ population using flow cytometry. *P<0.05 as determined by the one-way ANOVA with Bonferroni’s multiple comparison. (d) Representative photomicrograph of WT and C3−/− mouse lung tissue exposed to 6 months of smoke or air immunostained for expression of C3aR (green) or nuclei (blue; DAPI). Green arrows indicate C3aR+ cells. (e) to (h) Mouse BMDCs (2 × 105) were treated with C3aR agonist (CAS 944997-60-8; 20 ng/ml) or vehicle (2% DMSO) for 48 h. Expression level of C3aR1, Il6, Mmp9, and Mmp12 mRNA were measured using qPCR (n=4 in each group; **P<0.01 and ***P<0.001 as determined by student t test). (i) Human CD1a+ lung mDCs (2 × 105) were treated with purified human C3a (40 ng/ml) for 24 h or vehicle (media). Expression level of C3AR1 mRNA was measured by qPCR. n=3; **P<0.01 as determined by student t test. All gene expressions were normalized to 18S ribosomal RNA expression and analyzed by ΔΔCt. Results are represented as mean±s.e.m. from three independent experiments with 4–5 mice in each group (a–d). ANOVA, analysis of variance; BMDCs, bone marrow-derived dendritic cells; C3, complement protein 3; C3aR, C3a receptor; DAPI, 4',6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; mDCs, myeloid dendritic cells; MFI, mean fluorescent intensity; qPCR, quantitative reverse transcription PCR; WT, wild type. Scale bar: 20 μm.
