Degradation and inactivation of plasma tumor necrosis factor-alpha by pancreatic proteases in experimental acute pancreatitis.

BACKGROUND

Release of TNFalpha is thought to play an important role in mediating systemic effects in acute pancreatitis (AP). We have been unable to find an elevation of plasma TNFalpha in AP and hypothesize that it is susceptible to catabolism by circulating pancreatic proteases.

METHODS

(1) AP was induced in Sprague-Dawley rats by cerulein hyperstimulation preceded by intraductal infusion of saline (mild) or glycodeoxycholic acid (severe). Healthy and sham-operated animals served as controls. Severity of pancreatitis was confirmed by histology. Plasma TNFalpha levels were measured at various time points after induction of AP with competitive ELISA. (2) Recombinant rat TNFalpha (rrTNFalpha) was incubated with trypsin, elastase, chymotrypsin and pepsin. Western Blot was performed to visualize TNF degradation. (3) RrTNFalpha was incubated in a concentration and time-dependant manner with proteases and TNF bioactivity was evaluated with a cytotoxicity assay.

RESULTS

(1) Plasma TNFalpha levels in severe pancreatitis were significantly lower than in sham-operated controls after 0.5 and 6 h. (2) Incubation with proteases showed degradation in the presence of trypsin, elastase and chymotrypsin and no effect of pepsin. (3) There was a concentration dependent inactivation of rrTNFalpha in the presence of pancreatic proteases and a complete time-dependent inactivation in the presence of trypsin.

CONCLUSIONS

Plasma TNFalpha does not rise in experimental AP, and levels are significantly lower in severe pancreatitis compared to sham-operated controls. Our study demonstrates degradation and inactivation of TNFalpha by pancreatic proteases, suggesting that it is unlikely it plays an important role in the development of distant organ failure.