Pancreatic proteases in serum induce leukocyte-endothelial adhesion and pancreatic microcirculatory failure.
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 hypothesis of the study was that trypsin and elastase in serum alone lead to the expression of these complementary adhesion molecules and result in increased leukocyte-endothelial interaction (LEI). In addition we evaluated the preventative benefit of protease inhibition on these mechanisms.


METHODS
In vitro: Cultured endothelial cells (HUVEC) and human leukocytes (PMN) were stimulated with increasing doses of trypsin and elastase. In addition, pre-treatment of PMN or HUVEC was performed with protease inhibitors (Nafamostat mesilate, FUT and gabexate mesilate, FOY). The expression of ICAM-1 or MAC-1 was evaluated by flow cytometry. In vivo: Severe pancreatitis was induced in rats. Microcirculatory disturbances were evaluated by real-time confocal microscopy at 9 h in controls and acute pancreatitis with or without anti-protease treatment. Additionally, the effect of continuous trypsin and elastase infusion on pancreatic microcirculation and LEI were evaluated by intravital fluorescence videomicroscopy.


RESULTS
Up-regulation of MAC-1 and ICAM-1 expression requires the presence of serum. The maximal increase of MAC-1 and ICAM-1 expression was found at concentrations of trypsin or elastase characteristic for acute pancreatitis. FUT or FOY significantly reduced protease-induced expression of MAC-1 and ICAM-1. Real-time in-vivo microscopy revealed that functional capillary density in acute pancreatitis was significantly reduced (267.1 +/- 2.95/mm2 vs. 91.29 +/- 12.81/mm2) and treatment with FUT significantly reduced this effect (134.6 +/- 4.6/mm2; p < 0.05 vs. untreated pancreatitis). Infusion of trypsin or elastase alone increased LEI in vivo and reduced pancreatic perfusion.


CONCLUSIONS
Both trypsin and elastase up-regulate the expression of adhesion molecules on leukocytes and endothelial cells in the presence of serum. Increased LEI and reduced perfusion of the pancreas, characteristic of acute pancreatitis, is induced in vivo by infusion of pancreatic proteases and this effect is partially abrogated by their inhibitors. These results support the role of circulating trypsin and elastase in promoting pancreatic microcirculatory failure in experimental acute pancreatitis.
Keck
Tobias
T
Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.
Friebe
Veronika
V
Warshaw
Andrew L
AL
Antoniu
Bozena A
BA
Waneck
Gerald
G
Benz
Stefan
S
Hopt
Ulrich T
UT
Fernández-del-Castillo
Carlos
C
D000818:Animals; D002448:Cell Adhesion / Q000276:immunology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D008833:Microcirculation / Q000502:physiology; D009504:Neutrophils / Q000166:cytology* / Q000378:metabolism; D046790:Pancreas, Exocrine / Q000201:enzymology*; D010196:Pancreatic Elastase / Q000097:blood*; D019283:Pancreatitis, Acute Necrotizing / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D051381:Rats; D017207:Rats, Sprague-Dawley; D014357:Trypsin / Q000097:blood*
D010196:Pancreatic Elastase; D014357:Trypsin