Monitoring Fpr2 gene promoter activity and localization in Fpr2-/- mice bearing an in-frame GFP construct.

Stefania Bena, Vincenzo Brancaleone, Rod Flower, Mauro Perretti. William Harvey Reseach Institute, Bart’s and The London SMD, London, UK

The G protein coupled receptor (GPCR) formyl-peptide receptor type 2 or FPR2/ALX is one of the effectors of endogenous anti-inflammation1. GPCR induction could be a novel – much understudied - feature of an inflammatory reaction; thus we test here whether the degree of expression of this receptor changed during on-going inflammation. To do so, we have taken advantage of an Fpr2 (orthologue of human FPR2/ALX) mouse colony with an in-frame green fluorescence protein (GFP) gene ‘knocked-in’. Here we report the initial validation of protocols to monitor GFP expression for Fpr2 gene promoter activity.

Male Fpr2-/- and littermate controls (WT), 25±5 g body weight, were used for the study. Tissues and cells were harvested and GFP expression monitored by using fluorescence microscopy (tissue samples) and flow cytometry (cell samples). As a positive control, immunocytochemistry with a rabbit anti-mouse Fpr2 antibody (SantaCruz) was also performed on WT samples. GFP expression in cells (neutrophils, macrophages) was analyzed by flow cytometry using the FL-1 channel for the natural fluorescence; in some cases, a protocol for intracellular staining with an anti-GFP antibody (Invitrogen, A10262) was performed. Experiments were conducted with at least 3 mouse tissue or cell preparations.

Quantification of GFP fluorescence revealed that Fpr2 gene is active (or expressed by a higher number of positive cells) in the kidney (45% of fluorescence intensity - in compare with WT mice), followed by the aorta (35%), heart (30%), lung (15%), liver (10%) and spleen (10%). These data were validated by immunohistochemistry staining for Fpr2 using WT kidney and lung tissue section, confirming higher staining in the kidney (predominantly at the endothelium level and mesangial cells). For cellular analyses, Fpr2-/- blood neutrophils displayed ∼5% of GFP +ve events by flow cytometry (raising to 20% after anti-GFP antibody staining). Bone marrow cells showed instead ∼10% of GFP positive cells (≥20%, anti-GFP antibody). Resting peritoneal cells exhibited 5% of GFP positive cells, with a much higher degree of positive events (∼50%) following anti-GFP antibody staining.

We have monitored the pattern of expression of GFP as an indication of Fpr2 gene activity in resting organs and cells, developing immunological and imaging protocols aimed to determine, in a quantitative manner, GFP expression level. Next we will aim to use these protocols to study Fpr2 modulation during on-going experimental inflammation as a novel key step to unveil Fpr2 biology and, potentially, favour exploitation for drug discovery programmes.

1. Ye RD, Boulay F, Wang JM, Dahlgren C, Gerard C, Parmentier M, Serhan CN, Murphy PM. (2009) International Union of Basic and Clinical Pharmacology. LXXIII. Nomenclature for the formyl peptide receptor (FPR) family. Pharmacol Rev. 61(2):119-61.

Funded by the Wellcome Trust (programme grant 086867/Z/08/Z).