The expression of cellular adhesion molecules is upregulated and co-localized with the oxidative stress in the early course of experimental acute pancreatitis

Abstract

Using a novel method to demonstrate histologically the production of oxygen free radicals (OFR-s), this study investigates the possible role of cellular adhesion molecules (CAM-s) -in relation to the oxidative stressduring the early course of acute necrotizing pancreatitis (ANP). Similarly to other inflammatory pathologies, injury to the pancreatic acini supposedly results in the expression/upregulation of CAM-s on the periacinar endothelial cells as well as release of proinflammatory mediators. The adherent lenkocytes, mostly neutrophils (PMN), become activated and release OFR-s, microbicidal enzymes and more mediators to strengthen the inflammatory response. An uncontrolled inflammatory cascade may become locally and systemically deleterious. We have developed a novel technique to detect the presence and localization of OFR-s in vivo, using the cerium capture method. Reaction of OFR-s with CeC13 leads to cerium-perhydroxide precipitation. These deposits can be detected histologically by their laser reflecting properties using confocal laser scanning microscopy (CLSM) in the reflectance mode (RM). METHODS: ANP was induced in Wistar rats by retrograde infusion of taurocholate into the pancreatic duct. At different time points (1, 2, 24h) the animals were re-laparotomized and perfused with CeC13 (20mM in Hartmann solution) through the abdominal aorta, then sacrificed. Normal rats received a CeCI 3 perfusion in the same fashion and were used as controls. Pancreata were snap frozen in liquid nitrogen. Indirect immunofluorescence (FITC fluorochrome) was performed on serial frozen sections to label E-selectin, P-selectin, ICAM-1, VCAM-1, and PECAM molecules. Nuclei were colored by propidium-iodide. Simultaneous observation of CAM expression, cerium reflectance and cell type definition was performed by CLSM using multichannel detection. RESULTS: The capillaries in the pancreata of normal animals showed weak, sparse P-selectin and ICAM-1 positivity, with a constitutive expression of PECAM. Reflectance signals were negligible. At the early time points (1, 2h) of pancreatitis the tissue architecture was found to be relatively well preserved, an increase in endothelial P-selectin, and ICAM-1 immunoreactivity was observed. Strong, shining reflectance could be detected in the pancreatic microvasculature, as well as cloud-like signals over certain groups of acini. Numerous leukocytes adherent to CAM positive capillary walls were seen, however they presented small, focal, mostly intracellular reflectance signals, which were often localized at the contact points between the CAM-s and leukocytes. At 24h the samples were characterized by intense PMN infiltration, heterogeneous necrosis, strong, mostly perinecrotic P-selectin, and ICAM-1, but moderately increased E-selectin, and VCAM-1 expression. Large numbers of adherent, or already transmigrated PMN-s showed abundant intraand pericellular reflectance signals. CONCLUSION: Pancreatic endothelial CAM-s are upregulated early in ANP, and may play a role -in addition to cytokinesin the activation of adhering leukocytes. At the earliest time points the major source of OFR seems to be the pancreatic cell (xanthine-oxidase), later the activated PMN-s, thereby possibly contributing to local and distant organ damage. The CAM over-expression showed a significant spatial co-localization with the oxidative stress. The work of G. Telek was supported by a grant from IRMAD Foundation, France.