Trypsin stimulates production of cytokines from peritoneal macrophages in vitro and in vivo

Acute pancreatitis (AP) is characterized by release of proteolytic enzymes from the pancreas and a powerful inflammatory cytokine cascade that mediates the systemic manifestations and contributes to the mortality of the disease. The purpose of this study was to examine a potential link between pancreatic proteolytic enzymes, which are increased in AP, and cytokine production. To evaluate this, we incubated rat peritoneal macrophages (PMO) with increasing concentrations of trypsin and measured cytokine production. Supernatants from the cell cultures were assayed for TNF-alpha and IL-1 beta, and the PMO were collected for the evaluation of cytokine mRNA by polymerase chain reaction (PCR). Further to evaluate the role of pancreatic proteases in triggering the cytokine cascade in AP, trypsin was injected into the peritoneal cavity of Sprague-Dawley rats, and the production of cytokines was measured in the peritoneal fluid. Controls included injection of inactivated trypsin. Incubation of PMO with trypsin in vitro resulted in a dose-dependent increase in TNF-alpha production with maximal response (2,660.5 +/- 748.8 pg/mL) at 10 mu g/mL protease. Peak TNF-alpha and IL-1 beta release was noted 16 h after stimulation of the PMO (2,759.5 +/- 698.0 pg/mL and 160,596 +/- 4,065 cpm, respectively). Trypsin-induced TNF-a production was not due to release of cell-associated cytokine, inasmuch as activation of PMO with this protease causing an increase in TNF-alpha mRNA by 30 minutes, reaching a 14-fold increase at 4 h. Trypsin-injected animals produced TNF-alpha-containing ascitic fluid in a dose-dependent manner with peak TNF-alpha at 2 h (371.3 +/- 180 pg/mL) versus control (53.8 +/- 11.2 pg/mL; p < 0.022). No TNF-alpha was found in ascites of rats injected with heat-inactivated trypsin. Histologic examination of trypsin-injected animals revealed evidence of pulmonary inflammation at 2 and 4 hours. We conclude that the proteolytic enzyme trypsin stimulates cytokine production from macrophages in vitro and in vivo. This model demonstrates for the first time that trypsin is a potential mediator of the cytokine response seen during AP.