LIPOXIN A4 IMPROVES EFFEROCYTOSIS VIA INHIBITION OF THE HMGB1 IN HUMAN ALVEOLAR MACROPHAGES

Introduction Effective clearance of apoptotic cells by macrophages, termed efferocytosis, is a pre-requisite for successful resolution of inflammation. High mobility group box protein 1 (HMGB1), is an alarmin that may promote inflammation as well as suppress phagocytosis. Lipoxin A 4 , represents one of a unique class of lipid mediators that possess a wide spectrum of anti-inflammatory and pro-resolution actions. We hypothesised that lipoxin A 4 may promote both apoptosis in neutrophils, and stimulate macrophage efferocytosis, acting as an antagonist to HMGB-1. Methods Neutrophils were obtained from healthy volunteers and cultured for 24 h with or without lipoxin A 4. Apoptosis of neutrophils was determined with Annexin V/SyTox staining by flow cytometry. HMGB-1 levels in Acute Respiratory Distress Syndrome (ARDS) bronchoalveolar lavage fluid (BALF) was measured by ELISA. The effects of HMGB-1 and lipoxin A 4 upon alveolar macrophage efferocytosis was assessed by measuring the ingestion of CMFDA labelled apoptotic neutrophils by flow cytometry. The PI3K (P85) protein expression was measured by western blotting. Results Treatment of lipoxin A4 (100 nM) increased the apoptosis of neutrophils (p = 0.0244), and reduced the dead (p = 0.0238) and necrotic neutrophils (p = 0.0358) compared to control (n = 8). BALF from patients with ARDS suppressed efferocytosis of apoptotic neutrophils. The effects of BALF correlated with HMGB-1 levels in the BALF fluid. HMGB-1 decreased efferocytosis (p Conclusions Lipxin A 4 in vitro promotes the apoptosis but not necrosis of neutrophils. In tandem it stimulates efferocytosis of alveolar macrophages. Elevated HMGB-1 in ARDS BALF suppresses efferocytosis. Lipoxin A 4 can block these effects of HMGB-1. The effect of lipoxin A4 increasing efferocytosis was through ALX–PI3K signalling pathways. Lipoxin A 4 may therefore have potential as a therapeutic agent to promote the resolution of neutrophilic inflammation in ARDS.