Roles of endotoxin-related signaling molecules in the progression of acute necrotizing pancreatitis in mice.

OBJECTIVE: To study the potential roles of lipopolysaccharide (LPS) signaling molecules, LPS-binding protein (LBP), CD14, and Toll-like receptor 4 (TLR4) in mice with acute necrotizing pancreatitis (ANP).

METHODS: The ANP model was made by 7 intraperitoneal injections of cerulein (50 mug/kg) at hourly intervals, challenged by LPS administration of a dose of 5 mg/kg intraperitoneally 5 hours after the first injection of cerulein. Fifty-nine Balb/C mice were divided into 4 groups: group A, ANP control group, n = 18, received physiological saline; group B, anti-LBP group, n = 18, received 200 mug anti-LBP antibody; group C, anti-CD14 group, n = 18, received 20 microg anti-CD14 antibody; group D, anti-TLR4 group, n = 5, received 20 mug anti-TLR4 antibody. All treatments were given at 15 minutes before LPS injection. Serum amylase and lactic dehydrogenase (LDH) were measured. Histologic alteration of the pancreas was evaluated. Expressions of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and intercellular adhesion molecular (ICAM-1) mRNA were determined using reverse transcription polymerase chain reaction (RT-PCR). Myeloperoxidase (MPO) activity in pancreas was also analyzed. Nuclear factor-kappaB (NF-kappaB) p65 subunit was measured by immunohistochemistry and Western blotting.

RESULTS: Pretreatment of animals with anti-CD14 antibody resulted in a significant decrease in serum amylase and LDH levels, reduction of the severity of pancreatic injury, down-regulation of TNF-alpha, IL-1beta, and ICAM-1 mRNA expression, decrease in pancreatic MPO activity, and decrease in NF-kappaB expression compared with ANP control mice. In contrast, mice pretreated with anti-LBP antibody or anti-TLR4 antibody resulted in increasing of serum amylase and LDH levels, aggravation of the severity of necrosis and inflammation in pancreas, up-regulation of TNF-alpha, IL-1beta, and ICAM-1 mRNA expression, increasing of pancreatic MPO activity, and up-regulation of NF-kappaB expression compared with ANP control mice.

CONCLUSIONS: (1) LBP per se possesses a protective effect on ANP. It could facilitate clearance of endotoxin. (2) CD14 plays a crucial intermediate role in the progression of ANP. (3) TLR4, the essential transducer of LPS responses, may act independently of LPS. The antibody against TLR4 may mimic endotoxin and induce activation of downstream cytokines. For implication, recombinant LBP, anti-CD14 antibody, or silent TLR4 might alleviate the progression of ANP.