title: Pancreatic enzymes generate cytotoxic mediators in the intestine.
abstract: Recent evidence indicates that shock is accompanied by a failure of the mucosal barrier in the intestine and entry of pancreatic digestive enzymes into the wall of the intestine. To investigate the formation of cytotoxic mediators produced by enzymatic digestion of the intestine, we applied homogenates of rat small intestinal wall to human neutrophils and used flow cytometry measurements of propidium iodide uptake to determine cytotoxicity. We show that homogenates of the small intestine after ischemia by occlusion of the superior mesenteric and celiac arteries for 3 h, but not without ischemia, are cytotoxic. Digestion of homogenates of nonischemic intestinal wall with purified trypsin, chymotrypsin, or elastase, proteases normally present in the intestinal lumen, yielded cytotoxic mediators. Before cell death, we saw cell damage in the form of bleb formation and flow cytometry measurements of cell size changes due to blebbing. Cytotoxicity was prevented by serine protease inhibition with phenylmethylsulfonyl fluoride (PMSF) before, but not after proteolytic digestion of the wall homogenates, indicating that enzymatic action of proteases on the homogenate is necessary for cytotoxicity. Cytotoxicity of wall homogenates digested by enzymes in the fluid collected from the lumen of the intestine was greater than digests by the individual purified proteases. Cytotoxicity is undetectable if digestive enzymes in the luminal fluid are inhibited with a combination of enzyme inhibitors PMSF and 6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate before addition of wall homogenates. Passage of digested intestinal wall homogenates across a hydrophobic glass-fiber filter reduced cytotoxicity. Furthermore, we found that luminal fluid itself may be cytotoxic, possibly because of digestion of ingested food. To test whether digested food can be cytotoxic, we homogenized rat food and digested it in vitro with chymotrypsin or endogenous enzymes in luminal fluid. Cytotoxicity was significantly increased after digestion of food by luminal fluid compared with luminal fluid or undigested food. These results indicate the presence of a previously unknown mechanism for hemorrhagic necrosis in shock.
authors.affiliation: Department of Bioengineering, Whitaker Institute of Biomedical Engineering, University of California-San Diego, La Jolla, CA 92093-0412, USA. apenn@bioeng.ucsd.edu
authors.forename: Alexander H
authors.initials: AH
authors.lastname: Penn
authors.forename: Tony E
authors.initials: TE
authors.lastname: Hugli
authors.forename: Geert W
authors.initials: GW
authors.lastname: Schmid-Schönbein
mesh_terms: D000818:Animals; D017209:Apoptosis; D002918:Chymotrypsin / Q000737:chemistry / Q000378:metabolism; D003603:Cytotoxins / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007249:Inflammation; D007422:Intestines / Q000201:enzymology*; D007511:Ischemia / Q000378:metabolism; D008297:Male; D009504:Neutrophils / Q000378:metabolism; D010179:Pancreas / Q000201:enzymology*; D051381:Rats; D017208:Rats, Wistar; D012769:Shock
chemical_list: D003603:Cytotoxins; D002918:Chymotrypsin