Ischemia-reperfusion of the intestine produces a set of inflammatory mediators, the origin of which has recently been shown to involve pancreatic digestive enzymes. Matrix metalloproteinase-9 (MMP-9) participates in a variety of inflammatory processes including myocardial, hepatic, and pancreatic ischemiareperfusion. In the present study, we explore the role of neutrophil-derived MMP-9 in acute intestinal ischemia-reperfusion and its interaction with pancreatic trypsin. Male Sprague-Dawley rats were subjected to 45 minutes of superior mesenteric arterial occlusion followed by 90 minutes of reperfusion. In situ zymography of the proximal jejunum reveals increased gelatinase activity in the intestinal wall after ischemiareperfusion. Gel electrophoresis zymography and immunofluorescence co-localization suggests that this gelatinase activity is derived from MMP-9 released from infiltrating neutrophils. The role of intraluminal trypsin in this process was investigated using an in vivo isolated jejunal loop model of intestinal ischemia-reperfusion. Trypsin increased the inflammatory response after reperfusion, with an augmented neutrophil infiltration of the intestinal wall. Furthermore, trypsin stimulated a rapid conversion of neutrophil-released proMMP-9 into the lower molecular weight enzymatically active MMP-9. This process represents a powerful in vivo pathophysiological mechanism for trypsin-induced MMP-9 activation and is likely to play a central role in the development of acute intestinal inflammation and shock. Intestinal ischemia-reperfusion (I/R) is a localized pathology that often leads to multi-organ dysfunction and shock.

IntroductionRheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by sustained synovitis. Recently, several studies have proposed neutrophils and Th17 cells as key players in the onset and perpetuation of this disease. The main goal of this work was to determine whether cytokines driving neutrophil and Th17 activation are dysregulated in very early rheumatoid arthritis patients with less than 6 weeks of disease duration and before treatment (VERA).MethodsCytokines related to neutrophil and Th17 activation were quantified in the serum of VERA and established RA patients and compared with other very early arthritis (VEA) and healthy controls. Synovial fluid (SF) from RA and osteoarthritis (OA) patients was also analyzed.ResultsVERA patients had increased serum levels of cytokines promoting Th17 polarization (IL-1β and IL-6), as well as IL-8 and Th17-derived cytokines (IL-17A and IL-22) known to induce neutrophil-mediated inflammation. In established RA this pattern is more evident within the SF. Early treatment with methotrexate or corticosteroids led to clinical improvement but without an impact on the cytokine pattern.ConclusionsVERA patients already display increased levels of cytokines related with Th17 polarization and neutrophil recruitment and activation, a dysregulation also found in SF of established RA. 0 Thus, our data suggest that a cytokine-milieu favoring Th17 and neutrophil activity is an early event in RA pathogenesis.

Intestinal ischemia contributes to shock-induced multiple organ failure. Our recent evidence suggests that pancreatic proteases may be involved in the formation of inflammatory activators within an ischemic intestine. These inflammatory mediators are released early into the circulation and may contribute to the severe systemic inflammatory response syndrome (SIRS) during shock. We examined the impact of intra-intestinal pancreatic protease inhibition on acute intestinal ischemia-induced hypotension, the formation of activating factors for cardiovascular cells, as well as cremaster muscle cell death and intestinal injury by intravital microscopy. Male Wistar rats were divided into four groups: (1) a sham group; and experimental groups with 100 min of superior mesenteric artery occlusion (2) without (SMAO group), and (3) with intestinal lavage using Krebs-Henseleit solution (LAV group), or (4) lavage using the protease inhibitor gabexate mesilate in Krebs-Henseleit solution (FOY group, 0.37 mM). Intestinal ischemia and reperfusion-induced hypotension upon reperfusion was accompanied by a significant increase in the level of neutrophil-activating factors in the intestine and plasma. During reperfusion, a significant increase in leukocyte-endothelium interactions in postcapillary venules and parenchymal cell death were observed in the cremaster muscle in LAV and SMAO animals suggesting peripheral neutrophil cell activation. Intra-intestinal pancreatic protease inhibition resulted in a stable blood pressure throughout the experiment. Cell activation, leukocyte-endothelial interactions and cell death in the cremaster muscle were almost completely abolished in the FOY group. In addition, ischemia-induced intestinal mucosal injury was attenuated with intestinal pancreatic protease inhibition. These results indicate that intestinal pancreatic protease inhibition significantly attenuates intestinal ischemia-induced shock by reducing SIRS and gut injury.

Shock is associated with a dramatic rise in the level of inflammatory mediators found in plasma. The exact source of these mediators has remained uncertain. We recently examined a previously undescribed mechanism for production of inflammatory mediators in shock involving pancreatic digestive enzymes. The current in vitro study was designed to identify particular pancreatic enzymes and organs that may potentially produce inflammatory mediators. A selection of different organs from the rat (heart, liver, brain, spleen, pancreas, intestine, diaphragm, kidney, and lung) were homogenized and incubated with purified trypsin, chymotrypsin, elastase, lipase, nuclease, or amylase and the supernatant was incubated with fresh naïve leukocytes for 15 min. The level of leukocyte activation in the form of pseudopod formation and the fraction of cell death were measured. Without the addition of purified enzymes, only the homogenate of the pancreas yielded enhanced cell activation. Organs incubated with physiological concentrations of trypsin also stimulated significantly higher levels of pseudopod formation as compared with the undigested organs or enzymatic controls. Lipase and chymotrypsin were able to elicit cellular activation from selected organs such as the heart, intestine, liver and diaphragm. Undigested pancreatic homogenates were capable of producing substantial cell death, as compared with all other undigested organs. Intestinal digests with elastase, lipase, trypsin and chymotrypsin also stimulated significant cell mortality. Lipase-treated heart, liver, intestine, diaphragm, kidney, and lung stimulated cell death as well. We conclude that the intestine, as well as several other organs, may serve as a major source of inflammatory mediators during shock if exposed to digestive enzymes.