Pankreatische Proteasen im Serum sind Induktoren der endothelialen Leukozytenadhäsion und pankreatischen Mikrozirkulationsstörung

Abstract

Background: Neutrophil mediated tissue injury in acute pancreatitis includes a severe reduction of the functional microcirculation via interaction of adhesion molecules on leukocytes (Mac-1) and endothelium (ICAM-1). The aims of this study were (1.) to link the effect of trypsin and elastase to the expression of these complementary adhesion molecules (2.) to determine the effect on pancreatic microcirculation using intravital microscopy (3.) to evaluate the preventative benefit of protease inhibition on adhesion molecule up-regulation. Methods: In vitro: Cultured endothelial cells (HUVEC) and leukocytes (PMN) isolated from human whole blood were stimulated with increasing doses of trypsin and elastase. In addition pre-treatment of PMN or HUVEC was performed with the protease inhibitors nafamostat (FUT-175) and gabexate mesilate (FOY). The expression of ICAM-1 or Mac-1 was evaluated by flow cytometry. In vivo: Trypsin and elastase were infused to rats. Microcirculatory disturbances were evaluated by intravital microscopy (IVM) measuring sticking (adhesion > 30 sec) of PMN to the endothelium. Results: Mac-1 and ICAM-1 expression require co-stimulation with serum. The maximal increase of Mac-1 and ICAM-1 expression was found at concentrations of trypsin or elastase physiologic for acute pancreatitis. The inhibitors FUT-175 or FOY significantly reduced protease induced expression of Mac-1 and ICAM-1. Infusion of trypsin or elastase significantly increased adhesion of PMN to the endothelium (570.6 ± 16.5/mm2 and 459.5 ± 15.4/mm2) compared to controls (80.3 ± 9.8/mm2, p < 0.01). Conclusion: Both trypsin and elastase up-regulate the expression of adhesion molecules on leukocytes and endothelial cells in the presence of serum. This effect can be abrogated by protease inhibitors, with FUT-175 being more powerful than FOY. As demonstrated by intravital microscopy, infusion of trypsin and elastase alone significantly increase PMN adhesion in vivo. These results confirm the role of adhesion molecules in microcirculatory disturbances and suggest a successive deleterious pathway of free circulating trypsin and elastase, adhesion molecule expression and pancreatic microcirculatory failure.