Abstracts
1999 Digestive Disease Week

# 2201 SPECIFIC PANCREATIC ENZYMES ACTIVATE MACROPHAGES TO PRODUCE TNFa: THE ROLE OF NF-kB AND IkB PROTEINS
C Murphy, W Denham, G Carter, J Norman, Univ of South Florida, Tampa, FL

Introduction: The triggering event by which mononuclear cells throughout the body are induced to produce large amounts of cytokines during acute pancreatitis is unclear. In previous work investigating this issue, we have shown that three specific pancreatic enzymes (elastase, carboxypeptidase-A (carb-A), and lipase) induced dramatic TNFa protein from macrophages (Mf) while all others could not. This series of experiments was designed to examine the second messenger system by which this occurs. Methods. The rat Mf cell line, NR8383 was incubated for 3 hours with various proteases (elastase, carb-A, lipase, and trypsin) or LPS as a positive control. Activation of NF-kB was demonstrated by EMSA and presence of the inhibitory proteins IkBa and IkBb by western blot analysis. Production of TNFa mRNA was determined by RT-PCR. Results represent mean ± SEM with significance assigned to p < 0.05. Results. Elastase, carb-A, and lipase induced NF-kB activation, with specificity confirmed by competitive oligomer inhibition (Figure 1). Degradation of IkBb, not IkBa, was evident with these 3 enzymes and could be blocked by PDTC, an IkB phosphorylation inhibitor (data not shown). Consistent with our previous protein results, TNFa mRNA production was markedly increased by elastase, carb-A, and lipase (Figure 2), while also inhibited by PDTC. Trypsin was unable to elicit any of these responses. Conclusion: Macrophages can be induced by specific activated pancreatic enzymes, elastase, lipase, and carboxypeptidase-A, to produce TNFa. This process is dependent upon IkBb degradation and NF-kB activation, suggesting that these enzymes trigger this second messenger system through

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