Platelet-activating factor and protease-activated receptor 2 cooperate to promote lung inflammation through nuclear factor-kappa B transactivation

Objective: This study was conducted to investigate the role of protease-activated receptor 2 (PAR2) in platelet-activating factor (PAF)-induced lung inflammation and neutrophil recruitment in lungs of BALB/c mice. Methods BALB/c mice were pretreated with the PAR2 antagonist ENMD1068, PAF receptor (PAFR) antagonist WEB2086, or aprotinin, a reversible inhibitor of serine proteases, prior to intranasal instillation of carbamyl-PAF (C-PAF) or the PAR2 agonist peptide SLIGRL-NH2 (PAR2-AP). Leukocyte infiltration in bronchoalveolar lavage fluid (BALF), C-X-C motif ligand 1 (CXCL1) and CXCL2 chemokines, myeloperoxidase (MPO), and N-acetyl-glycosaminidase (NAG) levels in BALF, or lung inflammation were evaluated. Intracellular calcium signaling, PAFR/PAR2 physical interaction, and the expression of PAR2 and nuclear factor-kappa B (NF-КB, p65) transcription factor were investigated in RAW 264.7 cells stimulated with C-PAF in the presence or absence of ENMD1068. Results C-PAF- or PAR2-AP-induced neutrophil recruitment into lungs was inhibited in mice pretreated with ENMD1068 and aprotinin or WEB2086, respectively. PAR2 blockade impaired C-PAF-induced neutrophil rolling and adhesion, lung inflammation, and production of MPO, NAG, CXCL1, and CXCL2 production in lungs of mice. PAFR activation reduced PAR2 expression and physical interaction of PAR2 and PAFR; co-activation is required for PAFR/PAR2 physical interaction. PAR2 blockade impaired C-PAF-induced calcium signal and NF-κB p65 translocation in RAW 264.7 murine macrophages. Conclusion This study provides the first evidence for a cooperation between PAFR and PAR2 mediating neutrophil recruitment, lung inflammation, and macrophage activation.