OC 19.5 - Protease-Nexin-1,a serpin participating in neutrophil recruitment through CD11a integrin regulation.

Background: Inflammation and coagulation are two closely linked processes essential in the defense of the body.Thrombin,a key serine protease in the coagulation-cascade,is also involved in the inflammatory reaction.The activity of thrombin is mainly regulated by proteins belonging to the serpin superfamily.Among them,Protease-Nexin-1(PN-1)is the most effective tissue thrombin inhibitor.

Aims: Our aim is to understand how PN1 affects neutrophil functions.

Methods: PN1expression was analysed by immunocytochemistry and western blot on resting orLPS-activated neutrophils.We analyzed in vivo by intravital-microscopy the intravascular recruitment of neutrophils after inducing inflammation by topical deposition of LTB4 on mesenteric-veins.We also analyzed the inflammation in vivo using models of peritonitis induced by LPS/thioglycolate.We compared by flow-cytometry the production of ROS and the expression of the receptors expressed by WT/PN-1deficient neutrophils.Neutrophil adhesion on ICAM-1 matrix was studied.

Results: We showed that PN-1 is secreted by neutrophils after stimulation with LPS.We have observed a significantly reduced ROS-generation in PN-1-deficient neutrophils.In vivo models of peritonitis showed a decreased recruitment of PN-1-deficient neutrophils in the intraperitoneal lavages by 60%.We have demonstrated that the recruitment of neutrophils on inflamed mesenteric veins is less important in PN-1-KO mice.Since PN-1 is also expressed by platelets,we compared neutrophil recruitment in PF4-CRE + PN-1Flox/Flox mice(Platelet PN1deficient mice)with the one observed in LY6G CRE + PN-1F/Fmice(Neutrophil PN-1deficient mice).No significant modification of neutrophil vascular recruitment was observed in mice exhibiting PN-1deficient platelets and WT neutrophils,whereas a significant decreased neutrophil recruitment was observed in mice exhibiting PN-1deficient neutrophils.We have also shown the significant decrease of PN-1deficient neutrophils adhesion on ICAM-1.We demonstrated by flow cytometry,a 2-fold lower Mean Fluorescence Intensity(MFI) for the integrin CD11a on neutrophils from PN-1-deficient mice compared with neutrophils from WT mice.Such a deficit may explain the reduced neutrophil recruitment observed in PN1KOmice.

Conclusion(s): Our results demonstrated that PN-1promotes neutrophil recruitment and activity and could play an important role in the inflammatory reaction.