Urokinase receptor deficiency reduces neutrophil recruitment in sepsis and ameliorates survival

GRAZ-01-21 Introduction: Symptoms of generalized inflammatory syndromes are related to polymorphonuclear neutrophil (PMN) adhesion to microvascular endothelium and their consecutive extravazation. The urokinase receptor (u-PAR) has recently been shown to be critically involved in the regulation of the activity of β-integrin function. The role of u-PAR in inflammation has not been fully elucidated. Methods: PMN were percoll-purified from hind limb bone marrow of wild-type (wt) or u-PAR-deficient (−/−) mice and fluorescently labelled. PMN were superfused in a parallel plate flow chamber with a shear rate of 400s−1 over resting or TNFα stimulated murine endothelial cells (ECs) grown on collagen coated coverslips. Firm adhesion in this system is mainly mediated by MAC-1/ICAM-1. PMN adhesion (/20 mm2 of the coverslip) is reported as percentage of wt-PMN adhesion to resting ECs. u-PAR−/− or wt mice were injected i.p. with 30 or 45 μg g−1 bodyweight E. coli endotoxin (LPS) and left ventricular performance (by transthoracic echocardiography), survival time and mortality were recorded. Results: Compared to resting ECs adhesion of WT PMN increased to 172 ± 34% after 6-h stimulation with 100 nM TNFα (n = 5, P < 0.01), while u-PAR−/− PMN completely failed to increase adhesion to activated endothelium (99 ± 26% vs. 73 ± 26%, n = 6/5, P = NS resting vs. TNFα and P < 0.01, u-PAR−/− -TNFα vs. wt-TNFα). 68% of the u-PAR−/− (n = 22) but only 33% of the wt mice (n = 21) survived an injection of 30 μg g−1 LPS i.p. When 45 μg LPS was injected, only a trend for decreased mortality was detected after 72 h (43% vs. 25% survival, u-PAR−/− vs. wt, n = 21/20) but a significantly prolonged time to death was observed (29 ± 5 h vs. 16 ± 6 h, u-PAR−/− vs. wt). Increased survival was not fully explained by differences in LV function as 90 min after LPS injection shortening fraction (69 ± 3% vs. 62 ± 2%, u-PAR vs. wt, n = 5, P < 0.05) and velocity of circumferential fibre shortening (Vcf) were similar or only marginally different between groups. Conclusions: u-PAR expression on PMN is required for adequate PMN adhesion to activated EC. Lack of u-PAR protects mice from endotoxin induced systemic inflammatory responses and death, which is only in part related to cardiac function. Regulation of u-PAR mediated activation of integrins could be a suitable target for reducing inflammatory responses.