Anti-inflammatory agents inhibit leukocyte accumulation and vascular leakage induced by trypsin and trypsin-digested serum in hamster cheek pouch.

In the present study we investigated the effect of nordihydroguaiaretic acid (NDGA), indomethacin, and cortisone on trypsin-induced acute inflammation in the hamster cheek pouch. Permeability changes, evaluated by fluorescence microscopy after injection of FITC-dextran (MW 150,000), induced by trypsin (2.5 microM) and trypsinated serum (2.5 microM) were significantly suppressed by pretreatment with NDGA (20 mg/kg) and indomethacin (20 mg/kg). Pretreatment with cortisone (40 mg/kg) reduced the permeability changes induced by trypsinated serum but had no significant effect on trypsin-induced leakages. Accumulation of polymorphonuclear leukocytes, as calculated by a whole tissue histological technique, induced by trypsin or trypsinated serum, was significantly reduced by pretreatment with cortisone, NDGA, or indomethacin. These results indicate a role of both cyclooxygenase and lipoxygenase products in trypsin-induced acute inflammation in the hamster cheek pouch.

Activation of the complement cascade by trypsin.

In twenty-three patients with acute pancreatitis, the plasma levels of immunoreactive trypsin were determined with a RIA method. The patients were divided into groups according to the severity of the disease. Ranson's criteria and the development of multisystem organ failure were used for the classification. Elevated plasma levels of immunoreactive trypsin were found in all groups after admittance. Incubation of fresh human serum and plasma with bovine trypsin in concentrations between 10(-6) and 10(-4) M at 20 degrees C activated the complement cascade. The anaphylatoxins C3a and C5a were determined with a RIA and the terminal complement complex (TCC) with an ELISA method. C3a and C5a were released and TCC was formed. The effect of trypsin on leukocyte activation was determined with a chemiluminescence technique. Trypsin dissolved in saline did not activate the leukocytes. However, serum digested by trypsin-activated leukocytes in a dose-dependent manner. The present study supports the theory that trypsin can activate complement components and results in formation of split products which have potent vascular, and leukocyte activating effects.

Trypsin-induced vascular permeability and leukocyte accumulation in hamster cheek pouch: the role of complement activation.

Trypsin-induced acute inflammation was studied in hamster cheek pouch using intravital microscopy, correlative histology, and electron microscopy. Vascular permeability changes were monitored with intravital fluoroscopy, after intravenous injection of FITC-dextran (Mw 150,000), by counting the number of FITC-dextran leakages around the vessels. The number of extravasated polymorphonuclear leukocytes (PMNLs) was calculated by a histological technique. A dose-dependent increase in the number of FITC-dextran leakages, as well as the number of accumulated PMNLs, was found when trypsin was locally deposited in concentrations of 0.25-2.5 microM (15 microliters during 5 min). Local deposition of autologous serum treated with trypsin at final concentrations of 0.25-2.5 microM caused an increase in vascular permeability as equally pronounced as that of pure trypsin, but only a moderate PMNL accumulation which was not dose dependent. Trypsin at a 25 microM concentration resulted in numerous microbleedings and cessation of flow. The electron microscopy demonstrated inflammatory events (PMNL adhesion, diapedesis, and interstitial infiltration) in all treatment groups but they were more pronounced after trypsin exposure. Trypsin did not cause disintegration, cellular lysis, or increased mast cell degranulation. The permeability changes induced by trypsin (2.5 microM) and trypsinated serum (2.5 microM) were significantly suppressed by the addition of the chelating agent potassium-EDTA to the reaction mixture, indicating a calcium- or magnesium-dependent mechanism. Pretreatment of the animals with cobra venom factor (CVF), by which the plasma C3 concentration was reduced to less than 10%, inhibited the vascular leakages almost completely. The trypsin-induced accumulation of PMNLs was significantly reduced by potassium-EDTA as well as by pretreatment with CVF (P less than 0.01). These findings indicate a central role of complement activation in trypsin-induced acute inflammation in the hamster cheek pouch.

Accumulation of anaphylatoxins and terminal complement complexes in inflammatory fluids.

The presence of anaphylatoxins (C3a and C5a) and terminal complement complexes (TCC) in different inflammatory fluids and plasma was studied in 33 patients. Anaphylatoxins were assayed using a radioimmunoassay technique, and the terminal complement complexes were determined by an ELISA method. Patients with peritoneal (n = 14), pleural (n = 7), pericardial (n = 6) or burn bullae fluid (n = 6) were studied. High C3a and TCC concentrations were found in all these fluids. Elevated C3a and TCC concentrations in inflammatory fluids were found not only in patients with elevated plasma C3a and TCC concentrations, but also in patients with normal plasma levels. No increases in C5a concentration were observed in pleural or burn bullae fluid. In one patient with pericarditis, and in subjects with acute pancreatitis with ascites, high C5a levels were found in the fluid. However, the high TCC concentration in the fluids suggests that C5a had been formed but was probably removed by leucocytes present in the fluid.

Anaphylatoxins and terminal complement complexes in pancreatitis. Evidence of complement activation in plasma and ascites fluid of patients with acute pancreatitis.

Complement activation has been proposed as a mediator of remote complications of acute pancreatitis. Thirty-seven patients with acute pancreatitis were studied with respect to the formation of anaphylatoxins (C3a/C3adesArg, C5a/C5adesArg) and terminal complement complexes (TCC) in plasma and ascites fluid. The patients were classified according to Ranson's criteria. Eighteen patients with moderate or severe pancreatitis had higher maximum plasma C3a/C3adesArg and TCC concentrations than 19 patients with mild pancreatitis. During convalescence, the concentrations had returned to normal. High concentrations of C5a/C5adesArg and TCC were also found in ascites and pancreatic cyst fluid, drawn from patients with moderate or severe pancreatitis. As the terminal complement pathway activation is involved in reactive lysis and anaphylatoxins increase vascular permeability, anemia and impaired respiration in these patients may be influenced by complement activation.

Anaphylatoxin generation in acute pancreatitis.

Fifty-one patients with elevated serum amylase and clinical signs of acute pancreatitis were studied prospectively. The concentrations of anaphylatoxins (C3a and C5a) were measured with a radioimmunoassay and the activity of their inactivator was determined. The pancreatitis was classified as mild, moderate, or severe according to Ranson's 11 signs, appearance of peritoneal fluid, and development of multisystem organ failure (MSOF). Plasma C3a and C5a concentrations were elevated during attacks of acute pancreatitis. Anaphylatoxin levels correlated with the severity of the disease (C3a, P less than 0.001; C5a, P less than 0.05). The highest and most persistent levels were found in the group with MSOF. C3a levels decreased rapidly during recovery. In patients with complications like abscess or pseudocyst, the C3a elevation persisted until adequate treatment was instituted. In this study, no significant changes of the inactivator levels were found, except at discharge when the inactivator level of the severe group was elevated compared to that of the moderate and mild groups (P less than 0.05).