Resolving inflammation in experimental stroke through members of the formyl peptide receptor family.

Helen Smith, Felicity Gavins. Centre for Neurosciences, Dept. of Medicine, Imperial College, London, W12 0NN, UK.

Blood reperfusion into an ischaemic region following stroke is essential for tissue preservation, yet associated inflammation can worsen recovery; leukocytes-endothelial (L-E) interactions allow leukocyte emigration into surrounding tissue where they release highly reactive components, compounding cell damage. Pharmacological reduction of L-E interactions may therefore help to prevent ischaemia/reperfusion (I/R)-induced injury in stroke.

Members of the formyl peptide receptor family (FPR; Fpr in mouse) of G-protein-coupled receptors provide an endogenous mechanism for anti-inflammatory/pro-resolving activity. We hypothesised that administration of FPR ligands in a mouse model of cerebral I/R would reduce L-E interactions; this would provide evidence for a stroke therapy targeting the FPR system.

A model of global cerebral I/R was developed in male C57BL/6 mice (21-25g) using bilateral common carotid artery occlusion (BCCAO; 5min) and reperfusion (40min or 2h), with animals under deep anaesthesia (pentobarbital sodium; 100mg/kg) and requiring no artificial respiration. The resulting cerebral L-E interactions were viewed through a cranial window using intravital fluorescence microscopy (IVM). Temperature was maintained at 37±0.5°C throughout and rhodamine 6G (100μl; 0.02%) was administered 5min prior to IVM for visualisation of leukocytes under fluorescence.

Mice were administered 100μl treatment i.v. at the start of reperfusion: vehicle (saline/saline+ethanol), 15-epi-LXA4 (lipid mediator; FPR2/ALXhuman/Fpr2mouse-selective agonist; 38μM, vehicle = saline+ethanol), Ac2-26 (AnnexinA1 pharmacophore; FPR agonist; 324μM; vehicle = saline), Boc2 (pan-FPR antagonist; 100µM), WRW4 (FPR2/ALX-selective antagonist; 0.5μM) or Ac2-26+Boc2 or WRW4. After 40min/2h, characteristic stages of L-E interactions were analysed: rolling leukocyte flux (mm-2min-1), rolling velocity (μmsec-1), and leukocyte adhesion (mm-2min-1) were counted over 100μm vessel and expressed as mean ±SEM by unpaired t test; n = 4-6 mice/group.

15-epi-LXA4 treatment reduced L-E interactions (p<0.05) in cerebral I/R vs. vehicle at 40min reperfusion (56.1±9.0 vs. 236.9±26.7 rolling cells mm-2min-1; 19.9±19.9 vs. 267.2±34.8 adherent cells mm-2min-1); 15-epi-LXA4–treatment groups at 40min in fact demonstrated L-E interactions equivalent to sham-operated animals (sham = 43.6±12.24 rolling cells mm-2min-1; 59.31±20.23 adherent cells mm-2min-1)—a 90-100% reduction. This effect was not sustained at 2h (15-epi-LXA4 = 242.7±112.5 vs. vehicle = 279.6±79.1 rolling cells mm-2min-1; 300.7±87.6 vs. 470.5±46.4 adherent cells mm-2min-1), unless treatment were administered 40min into reperfusion (28.5±4.0 vs. 132.6±29.1 rolling cells mm-2min-1; 143.2±16.2 vs. 219.4±118.6 adherent cells mm-2min-1; p<0.05) rather than at its start.

Ac2-26 treatment decreased adhesion (p<0.05) at 40min vs. vehicle (80.0±44.7 vs. 315.4±37.25 adherent cells mm-2min-1) and all L-E interactions at 2h (58.2±21.22 vs. 112.6±74.47 rolling cells mm-2min-1; 77.3±15.2 vs. 610.7±102.3 adherent cells mm-2min-1).

Combined treatment with Ac2-26 plus FPR antagonists Boc2 or WRW4 (FPR2/ALX-selective) abrogated the effects of Ac2-26 at 40min (adhesion = 454.2±78.43 and 326.2±50.2 cells mm-2min-1; p<0.05), suggesting an Fpr2 mechanism. These data, and Boc2/WRW4-only treatment groups, demonstrate a quadrupling of L-E interactions at 40min when these FPR antagonists are administered compared with vehicle-treatment groups, and a trend towards slower rolling velocities vs. other groups (WRW4+40min reperfusion = 14.4±2.8μmsec-1 vs. vehicle = 34.7±5.1μmsec-1).

Unexpectedly, abrogation was not sustained at 2h; co-treatment with antagonists abrogated anti-inflammatory effects of Ac2-26 minimally compared with the 40min time point. Most surprisingly, the co-treatment groups (as well as Boc2/WRW4-alone treatment groups) produced cell adhesion levels significantly lower than vehicle-treated groups (Ac2-26+Boc2/WRW4 vs. vehicle = 238.7±41.6/193.2±41.31 vs. 610.7±102.3 cells mm-2min-1; p<0.05).

We show that 15-epi-LXA4 and Ac2-26 both reduce L-E interactions in an experimental model of global I/R, that this activity is likely to occur through Fpr2 in our model, and that the effect is reduced or enhanced (respectively) as reperfusion progresses. These data suggest not only that FPRs could play a significant role in I/R injury following stroke and provide a therapeutic target, but that time of treatment administration has significant impact on efficacy.