2008 Landis Award lecture. Inflammation and the autodigestion hypothesis

Abstract

Although long recognized in microvascular research, an increasing body of evidence suggests that inflammatory markers are present in human diseases. Since the inflammatory cascade serves as a repair mechanism, the presence of inflammatory markers in patient groups has raised an important question about the mechanisms that initiate the inflammatory cascade (i.e., the mechanisms that cause tissue injury). Using a severe form of inflammation, shock, and multiorgan failure, for which there is no accepted injury mechanism, we summarize studies that suggest that the powerful pancreatic digestive enzymes play a central role in the destruction of the intestine and other tissues if their compartmentalization in the lumen of the intestine and in the pancreas is compromised. Further, we summarize evidence that uncontrolled degrading enzyme activity in plasma causes proteolytic cleavage of the extracellular domain of membrane receptors and loss of associated cell functions. For example, in a model of metabolic disease with type II diabetes, proteolytic cleavage of the insulin receptor causes the inability of insulin to signal glucose transport across membranes. The evidence suggests that uncontrolled proteolytic and lipolytic enzyme activity may trigger the mechanism for tissue injury. The significance of such mechanisms remain to be explored in human diseases.

Figure 1. Auto-digestion of the intestine by pancreatic enzymes during ischemia

Micrographs of rat intestinal wall morphology (semi-thin section stained with toluidine blue) and zymographic image with trypsin fluorescently quenched substrate (green fluorescence) before (Panels A, B, C, respectively) and after 45 min intestinal ischemia (D, E, F). Note the extensive damage to the microvilli and mucosal epithelium (D, E, arrows) and penetration of activated trypsin across the full thickness of the intestinal wall (F) with activation of trypsin activity (bright green fluorescence). Adapted from (29, 92).

Figure 2. Blockade of Digestive Enzymes Preserves Villi

Intestinal villi in the rat before (Panel A) and after 90 min shock by occlusion of the superior mesentery artery without (B) and with (C) serine proteases blockade with ANGD in the lumen (L) of the intestine. Length of crossbar 100 μm. Note a significant protection of the intestinal villi structure by blockade of luminal pancreatic proteases. Similar protection of villi is observed in severe porcine shock after blockade of pancreatic enzymes in the intestinal lumen. Adapted from (74).

Figure 3. Blockade of Digestive Enzymes Prevents Peripheral Inflammation

Micrograph of postcapillary venules in cremaster muscle at 80 min ischemia by superior mesentery artery occlusion (left column) followed by 80 min reperfusion. (Panels A, B) Non-ischemic control (sham), (C,D) with buffer as fluid for intestinal lavage, (E,F) with the serine protease inhibitor FOY (0.37 mM) in the lumen of the intestine (remote from the microvascular observation site). Note, the characteristic leukocyte adhesion to microvascular endothelium during shock in the post-capillary venules (arrows in panel D) is absent after blockade of the digestive enzymes in the lumen of the intestine (29). Bar equals 20 μm.

Figure 4. Enhanced MMP activity in the SHR Microcirculation

Digital fluorescent micrographs of WKY and SHR mesenteric microvessels labeled with fluorogenic peptide substrate showing matrix metalloproteinase (MMP-2, 9) enzymatic activity. Arterioles (A) and venules (V) are visible. Note the enhanced fluorescent emission over the endothelial cells and mast cells in the SHR, an affect that is less detectable after the doxycycline treatment. Adapted from (25). Bar equals 60 μm.

Figure 5. Elevated MMP-9 protein levels in SHR Tissue

Selected micrographs of microvessels and interstitium of WKY and SHR mesentery after MMP-9 immunolabeling (with Vector NovaRED substrate) (25). Note the pronounced labeling in SHR endothelial cells of arterioles (A) and venules (V) as well as in interstitial mast cells and fibroblasts (arrows).

Figure 6. Extracellular Insulin Receptor Cleavage and Insulin Resistance

Panel A: Typical micrographs of immunolabel (Vector NovaRed) for the extracellular domain binding site of the insulin receptor α on fresh leukocytes (neutrophils and monocytes) from WKY and SHR. Note the reduced density of the insulin binding sites on the SHR leukocytes associated with about 20% average reduction of the receptor density on the plasma membrane and with reduced transport of glucose (shown with fluorescence-tagged analog of glucose in panel B) into the cell cytoplasm (25). Panel C: Glucose transport into naïve leukocytes from the normotensive Wistar strain before and after a 30 min incubation in fresh plasma of the Wistar (Wistar and Wistar-W in panel C) and in plasma from WKY and SHR (Wistar-WKY and Wistar-SHR in panel C). For quantitative measurements see (25).

Figure 7

(A) Schematic Diagram of Autodigestion by Pancreatic Enzymes in the Intestine. The fully activated pancreatic digestive enzymes are during normal digestion contained within the lumen of the intestine by the mucosal epithelial barrier (Left Panel). Compromise of the mucosal barrier, e.g. due to ischemia or infection of the mucosal barrier, permits entry of fully activated pancreatic enzymes into the wall of the intestine (Right Panel). This creates two complications, generation if proinflammatory and cytotoxic mediators, and morphological destruction of the mucosal barrier allowing further entry of digestive enzymes into the wall of the intestine. Pancreatic enzymes as well as inflammatory mediators are carried out of the intestinal wall via the intestinal venous system, the intestinal lymphatics and by leakage across the outer coat of intestine connective tissue (serosa) into the peritoneum and into the central circulation. Appearance of pancreatic enzymes and these inflammatory mediators leads to central inflammation and innocent bystander organ damage. i.e. multi-organ failure. (B) Schematic Diagram of Protease Activity, Insulin Receptor Cleavage, and Insulin Resistance. The normal transmembane transport of glucose into the cell cytoplasm via the glucose transporter GLUT-4 as well as gene expression and growth factor synthesis requires intracellular signaling from the activated (phosphorylated) insulin receptor after binding of insulin to its extracellular domain (left panel). The appearance of uncontrolled degrading enzyme activity (e.g. due to MMPs, serine proteases) leads to cleavage of the extracellular domain of the insulin receptor (right panel), a lack of insulin binding sites on the insulin receptor, reduced intracellular signaling with attenuated glucose transport by GLUT-4, i.e. type II diabetes with insulin resistance due to reduced insulin receptor binding sites.