- Email: [email protected]
Pancreatic enzymes sustain systemic inflammation after an initial endotoxin challenge
Pancreatic enzymes sustain systemic inflammation after an initial endotoxin challenge Florian Fitzal, MD, Frank A. DeLano, MS, Corey Young, Henrique S. Rosario, MD, Wolfgang G. Junger, PhD, and Geert W. Schmid-Scho¨nbein, PhD, La Jolla, Calif; Vienna, Austria; and Lisbon, Portugal
Background. Sepsis is accompanied by severe inflammation whose mechanism remains uncertain. We recently demonstrated that pancreatic proteases in the ischemic intestine have the ability to generate powerful inflammatory mediators that can be detected in the portal vein and in the general circulation. This study was designed to examine several circulatory and inflammatory indices during experimental endotoxemia and intraintestinal pancreatic protease inhibition. Methods. Immediately after intravenous endotoxin administration, the small intestine was subjected to intraluminal lavage with and without gabexate mesilate, an inhibitor of pancreatic proteases. Shams and rats without lavage served as controls. Hemodynamics, leukocyte (neutrophil and monocyte), and endothelial cell activation, as well as organ injury in the intestine and the cremaster muscle, were examined. Results. After endotoxin administration, control rats developed hypotension, tachycardia, hyperventilation, and leukopenia. The intestine and plasma contained mediators that activated leukocytes. The leukocyte-endothelial interaction within the cremaster muscle microcirculation was enhanced. Endotoxin administration resulted in elevated interleukin-6 plasma levels. Histologic evidence indicated liver and intestinal injury. In contrast, blockade of pancreatic proteases in the intestinal lumen significantly improved hemodynamic parameters and reduced all indices of inflammation in plasma and cell injury in skeletal muscle microcirculation. Conclusions. Inflammatory mediators derived from the intestine by pancreatic proteases may be involved in the prolonged inflammatory response and sustain symptoms of sepsis after endotoxin challenge. (Surgery 2003;134:446-56.) From the Department of Bioengineering, the Whitaker Institute for Biomedical Engineering and Department of Surgery, UCSD Medical Center, University of California–San Diego, La Jolla, Calif; Department of Surgery, University of Vienna Medical School, and Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria; and Institute of Biochemistry, Faculty of Medicine of Lisbon, Lisbon, Portugal
SYSTEMIC INFLAMMATION (also denoted as systemic inflammatory response syndrome) and sepsis remain the leading causes of death in intensive care units. Evidence indicates that the severity of inflammation as measured by leukocyte (neutrophil and monocyte) activation in the circulation may serve as a predictor for survival,1 and that inThis work was supported by the Max Kade Foundation, and in part by NIH Grants HL-43026, HL-67825, GM-60475, and by FLAD 460/2000. Accepted for publication April 12, 2003. Reprint requests: Florian Fitzal, MD, Department of Surgery, University of Vienna Medical School, Waehringer Guertel 18-20, 1090 Vienna, Austria. Ó 2003, Mosby, Inc. All rights reserved. 0039-6060/2003/$30.00 + 0 doi:10.1067/S0039-6060(03)00168-5
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flammatory mediators are produced during sepsis. These inflammatory mediators are detectable in plasma and exhibit a variety of activities which, in addition to the activation of circulating leukocytes, include suppression of T lymphocyte proliferation and myocardial contraction, as well as several other cell and organ dysfunctions.2-4 The intestine appears to play a central role during sepsis.5,6 It generates different inflammatory mediators,7-10 some of which have been characterized (tumor necrosis factor, interleukins, leukotrienes, platelet activating factor), and some of which remain unidentified.9,11 Translocation of intestinal bacteria and endotoxin has been suggested to be responsible for the prolonged inflammation during sepsis. However, a number of attempts to relate intestinal-derived endotoxemia to mortality rates in clinical sepsis
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studies have failed so far.12,13 The intestine may generate factors that activate inflammatory cells even in the absence of endotoxin or bacteria.14-16 Thus, we may have to reexamine our basic hypothesis for the pathophysiology of sepsis because other inflammatory mediators besides endotoxin, cytokines, or lipid-derived mediators may produce cell activation and multiple organ injury.12,17 Recently we demonstrated an important role of pancreatic digestive enzymes during multiorgan failure following intestinal ischemia. After entry into the intestinal tissue because of ischemiainduced breakdown of the mucosal barrier, digestive enzymes may generate a set of powerful inflammatory mediators in the intestinal tissue that activate leukocytes and endothelial cells in the microcirculation.18,19 The mediators produced by the digestive enzymes enhance the oxidative stress, cause myocardial depression, and lead to apoptotic cell death in the tissue parenchyma and rapid mortality.11,20,21 Inhibition of pancreatic proteases in the lumen of the intestine significantly improves the hemodynamics and reduces inflammation during intestinal ischemia/reperfusion-induced shock.19,22,23 In endotoxin shock, however, the impact of the inflammatory mediators derived from pancreatic proteases on the cardiovascular system is unexplored. We hypothesize that sepsis-induced hypoperfusion of the intestine and elevated permeability of the mucosal epithelium may lead to translocation of pancreatic enzymes from the lumen into the wall of the intestine, followed by generation of factors that activate inflammatory cells within the wall. The inflammatory mediators may then enter the circulation and maintain and enhance inflammation during sepsis, even though it may have been triggered by endotoxin. We examine here one particular aspect of this hypothesis. After endotoxin administration, we study the effect of intraintestinal pancreatic protease inhibition (IPI) on central cardiopulmonary dynamics, systemic inflammation, the level of inflammatory mediators that cause leukocyte activation, and peripheral muscle parenchymal cell death. MATERIAL AND METHODS Twenty-five male Wistar rats (180-220 g; Charles River Breeding Laboratories, Wilmington, Mass) were maintained on a standard rat chow and water ad libitum. All experiments were reviewed and approved by the University of California San Diego Animal Subjects Committee. The rats were fasted overnight. After general anesthesia (60 mg/kg
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intramuscular pentobarbital), the left carotid artery and right jugular vein were cannulated (polyethylene tubing, PE-50, Clay Adams, Parsippany, NJ) to record arterial pressure and heart rate and to administer supplemental anesthetic agent or fluid support (Ringer’s lactate 2 mL/h), respectively. A tracheal tube was inserted to minimize respiratory complications. Escherichia coli-derived endotoxin (serotype 127:B8, Sigma Chemical Co, St. Louis, Mo; 4 mg/kg) was administered intravenously into the jugular vein over 5 min. Each animal was then observed during the subsequent 5-hour period. Experimental groups. The rats were divided randomly into 4 groups (each with 5 rats): 1. SHAM group: sham surgery without endotoxin administration or intestinal lavage 2. CON group: endotoxin administration without intestinal lavage 3. LAV group: endotoxin administration and intestinal lavage with 450 mL of Krebs-Henseleit buffer (see the following sections) 4. FOY group: endotoxin administration and intestinal lavage with 450 mL of Krebs-Henseleit buffer with 0.38 mmol/L of the protease inhibitor gabexate mesilate (FOY; Ono Pharmaceutical, Tokyo, Japan). Intestinal lavage and IPI. In the LAV and FOY groups, a polyethylene tube (PE 250, Clay Adams) was inserted into the duodenal lumen immediately distal to the pylorus (fixed with silk 4/0) and another tube was placed into the terminal ileum to permit free fluid discharge into a collection container. At the onset of endotoxin administration, the small intestinal lumen was rinsed gently (10 mL/min) with 100 mL of Krebs-Henseleit buffer solution (7.7 g NaCl, 0.35 g KCl, 0.29 g CaCl2, 0.3 g MgSO4 per liter of sterile water, 388C, pH 7.6-7.8) without (LAV group) or with FOY for intestinal protease blockade (FOY group). Also in the LAV and FOY groups, the small intestinal lumen was rinsed gently (10 mL/min) every 30 minutes with another 50 mL of buffer (with or without FOY, respectively). Pilot studies with intravital microscopy showed that intestinal lavage did not alter the intestinal microcirculation, either with or without FOY (unpublished data). Blood pressure dropped transiently in some rats during the first 5 minutes of lavage, leading to a short period (about 1 minute) of hypotension, with blood pressures of 60 mm Hg. We did not observe an increase in blood pressure resulting from intestinal lavage. In preliminary studies we examined endotoxin activity by the limulus
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amebocyte lysate activity test (unpublished results) in the intestinal fluid before and after intestinal lavage (n = 5 per group). Intestinal lavage with or without FOY did not influence endotoxin activity in the intestinal fluid when compared with prelavage levels (LAV group, prelavage mean ± SD: 140 ± 179 vs postlavage 86 ± 36 EU/mL endotoxin activity; FOY group, prelavage mean ± SD: 66 ± 72 vs postlavage 41 ± 20 EU/mL endotoxin activity). Cremaster muscle microcirculation. To examine leukocyte and endothelial cell activation in the peripheral microcirculation, the cremaster muscle was exposed for intravital microscopy24 at 120 minutes after endotoxin administration. The muscle preparation was continuously superfused with Krebs-Henseleit bicarbonate-buffered solution saturated with a mixture of 95% N2 and 5% CO2 at 378C. In each muscle, 1 terminal arteriole (30-50 lm diameter), 3 capillary observation fields (20 to 30 capillaries/field), and 3 postcapillary venules (2030 lm diameter) in the central portion of the muscle were examined with a 320 objective (numeric aperture = 0.5) for the capillary fields and the arterioles and venules with a 340 water-immersion objective (numeric aperture = 0.76). After a 30-minute adaptation period, the first microvascular bright field and fluorescent images were recorded with a color CCD camera (model VI470, Optronics, Goleta, Calif) and a videotape recorder (AG6300, Panasonic; Matsushita Electric Ind Co, Japan) for offline data analysis. Propidium iodide (Sigma) was added to the cremaster muscle superfusate (1 lmol/L final concentration) to label the nuclei of dead cells.25 At each time point, the images of the microvessels were recorded for 1 minute; otherwise, the tissue was not illuminated to minimize tissue injury because of the exposure of fluorescent light. Images of the muscle microcirculation were recorded 180, 240, and 300 minutes after endotoxin administration. Microvascular measurements. Red blood cell velocity (VRBC) was measured online by the crosscorrelation technique (Vista Electronics Company, Ramona, Calif). Vessel diameters and lengths as well as the number of rolling and adherent leukocytes in the pre-capillary arterioles and in postcapillary venules at each time point were determined. Leukocytes that were rolling continuously along the vessel wall were counted per unit time. Leukocytes attached firmly to the endothelium for a period of 30 seconds or longer were referred to as adherent cells. Leukocyte rolling velocity (VWBC) on the endothelium in postcapillary venules was measured off-
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line from the displacement of individual leukocytes from image frame to frame (time duration for 1 video frame = 1/30 sec). The leukocyte adhesion index (LAI) was calculated from the ratio of VWBC and VRBC in the same postcapillary venule segment measured during the same time interval. LAI falls between 0 and 1. A decline in LAI results from an increase in leukocyte-endothelial cell membrane attachment.26 In each microvascular field, functional capillary density was determined as the fraction of perfused capillaries and the number of all (perfused and nonperfused) capillaries. Tissue samples. Before intestinal lavage and at the end of the experiment, intestinal fluid was collected from the ileal tube and centrifuged at 500 g for 5 minutes. Aliquots were stored (708C) until use for measurement of protease activity (see the following section). One aliquot of blood (1 mL containing 10 U/mL heparin) was collected before endotoxin administration, and 1 aliquot at the end of the experiment. The samples were centrifuged (500 g for 5 minutes) and the plasma stored (708C) for measurement of leukocyte activation and IL-6 levels (see the following section). An additional 0.1 mL of heparinized blood was used to determine the leukocyte counts in peripheral blood (Unopette Microcollection System, Bekton, NJ). After euthanasia (5 hours after endotoxin administration), a segment of the ileum (5 cm) was excised, rinsed in phosphate buffered saline (PBS, 10 mmol/L) and fixed in 3% formalin (24 hours). The intestinal segments were embedded in resin, sectioned (1 lm section thickness), stained with toluidine blue, and assessed by light microscopy for morphologic damage (no quantification, 3 sections per animal). Another segment of the terminal ileum (1 g) was excised, and all intestinal contents were rinsed. The segment was placed into 3 mL of a PBS solution, homogenized, and centrifuged at 1000 g for 10 minutes. The supernatants from the homogenate were collected and stored (708C) for later measurements of leukocyte activation as pseudopod formation (see the following section). Protease activity assay. Total protease activity in intestinal fluid was measured with a protease assay kit (EnzChek, Molecular Probes, Eugene, Ore). The protease activities detected by this kit included elastase, trypsin, chymotrypsin and pepsin. The fluorescence intensity of the kit mixed with the samples was measured with a fluorometer and enzyme activity was expressed in fluorescence units (FU) with a lower detection limit between 1.0 3 103 and 4.4 3 105 FU.
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Leukocyte activation. Supernatant samples of the intestinal homogenates and plasma samples were mixed with naive human leukocytes (from asymptomatic volunteers). The inflammatory tripeptide F-Met-Leu-Phe (107 mol/L) and buffer served as positive and negative controls, respectively (not shown). Exposure of the leukocytes leads to general activation (eg, superoxide formation, degranulation, expression of adhesion molecules, polymerization of actin with pseudopod formation, and mechanical stiffening of the cytoplasm). Pseudopod formation leads to leukocyte entrapment in the microcirculation and migration into extravascular tissue. Thus, as an index of leukocyte activation, we determined the fraction of cells with pseudopod formation as described earlier.21 One hundred leukocytes (neutrophils and monocytes) were microscopically examined (1003 objective, numerical aperture 1.4, 103 objective) and cells with cytoplasmic projections greater than 1 lm were classified as activated leukocytes. IL-6 measurements. IL-6 levels of prediluted plasma samples (1:100) were measured with a commercial ELISA kit (Quantikine M ELISA; R&D Systems Inc., Minneapolis, Minn). Samples were measured in duplicates and values were expressed as ng/mL. IV administration of gabexate mesylate. During intestinal lavage, FOY may be partially absorbed into the circulation, where it can reduce cytokine levels,27 leukocyte activation,28 and oxygen-free radical formation,29 and therefore may influence parameters of septic shock. To investigate whether intestinal lavage with FOY affects the hemodynamics by way of intestinal protease inhibition or by direct intravascular systemic actions, 5 rats were prepared as described previously. Endotoxin administration (4 mg/kg) was followed by 8 FOY bolus administrations (10 mg/kg/h) into the jugular vein every 30 minutes (FOYiv group). This dose is sufficient to reduce leukocyte adhesion and attenuate ischemia/reperfusion injury.28 Hemodynamic parameters were measured as described previously. Statistical analysis. Data are presented as mean ± standard deviation. Repeated measurements of protease activity in intestinal fluids and the plasma levels of interleukin (IL)-6 were analyzed using the paired Student t test. The Mann-Whitney Rank Sum test was used to assess differences in trypsin activity between the 2 groups. Repeated measurements of microvascular and hemodynamic parameters in the same group were compared with repeated analysis of variance (ANOVA). For normally distributed data, ANOVA was followed by the Tukey’s multiple comparison correction test (in comparison with
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control baseline); for Poisson distributed data, the ANOVA on ranks was followed by Dunnett’s correction test. ANOVA was used to assess differences between groups for microcirculatory, hemodynamic, and IL-6 measurements as well as leukocyte activation at the same time. ANOVA was followed by Tukey’s correction test for normally distributed data, for Poisson distributed data, and ANOVA on ranks was followed by Student Newman Keuls’ correction test; P < .05 was considered to be significant with a power of .8. RESULTS Intraintestinal pancreatic protease inhibition (IPI). Intraintestinal lavage with FOY served to reduce intestinal serine protease activity (FOY group, 92 ± 17 units) compared with its baseline value (CON group, 2604 ± 734 units, P < .006) or with intestinal protease activity after lavage with vehicle (LAV group, 630 ± 441 units, P < .029). Hemodynamic parameters. Endotoxin administration in the CON and LAV groups was followed by a significant reduction in mean arterial blood pressure compared with the baseline and with the SHAM group. In contrast, after an initial drop at 20 minutes, the arterial blood pressure in the FOY group increased to pre-endotoxemia levels and remained stable thereafter. Throughout the rest of the experiment there were no significant differences in femoral pressure between SHAM, FOY, and their respective baseline levels. There were also no significant differences in pressure between the FOY and LAV groups (Fig 1, A). The FOY group exhibited a trend (not significant) toward higher arterial pressure than did the LAV group. After endotoxin administration, the heart rate (Fig 1, B) and respiratory rate (Fig 1, C) increased significantly in the CON, LAV, and FOY groups at 60 minutes when compared with the SHAM group. At 180 minutes after endotoxemia, the heart and respiratory rate of the LAV and CON group remained elevated significantly when compared with SHAMs. The FOY group had lower values than the CON group (P < .004), which were not significantly different from the SHAM values. At 300 minutes, the FOY group had lower heart and respiratory rates than the LAV and CON groups (P < .002). Leukocyte counts. Endotoxemia induced a significant leukopenia in venous blood of all groups at 300 minutes (CON: 2410 ± 650 cells/mm3; LAV: 1380 ± 620 cells/mm3; FOY: 3010 ± 2390 cells/ mm3) compared with the SHAM group (7400 ± 1610 cells/mm3; P < .001).
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Fig 1. Cardiopulmonary parameters in rats measured during 300 minutes after endotoxin administration (4 mg/kg intravenously). The groups are: SHAM, CON (controls with endotoxin administration), LAV (endotoxin administration and intestinal lavage with buffer), FOY (endotoxin administration and intestinal lavage with the protease inhibitor FOY, 0.37 mM). Hypotension (A), tachycardia (B), and hyperventilation (C) were attenuated by intraintestinal protease inhibition. Significant differences at P < .05 between LAV compared with its baseline (*); FOY compared with its baseline (y); CON compared with its baseline and SHAM (à); CON compared with its baseline (§); CON, LAV, FOY compared with SHAM (k); LAV compared with FOY (P < .002) (P); FOY compared with CON (P < .004) (#); and LAV compared with SHAM (P < .004) (**); n = 5 rats in each group.
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Fig 2. Systemic cell activation. Leukocyte and endothelial cell activation in the cremaster muscle in postcapillary venules measured by intravital microscopy at 180, 240, and 300 minutes after endotoxin administration. Groups are the same as in Fig 1. A, Number of rolling leukocytes. B, Number of adherent leukocytes to endothelial cells. C, Leukocyte adhesion index (LAI = VWBC/VRBC) as indicator for endothelial/leukocyte activation and interaction. D, The number of propidium iodide (PI) positive parenchymal cremaster cells as marker for peripheral cell death. Data are presented as mean ± SD. *P < .05 compared with SHAM at the same time point. yP < .05 FOY compared with CON and LAV at the same time point. àP < .05 when compared with 180 minutes after endotoxemia.
Microvascular leukocyte kinetics. The number of rolling leukocytes (Fig 2, A) in postcapillary venules of the CON and LAV groups at 240 minutes and 300 minutes (P < .004) after endotoxin administration were increased compared with the SHAM group. In the FOY group, the number of rolling leukocytes did not differ from that of the SHAMs. FOY attenuated the reduction of rolling leukocytes in the CON and LAV groups at 240 and 300 minutes after endotoxemia (P < .004). In the CON and LAV groups, the central leukopenia (see previous) was accompanied by an increased number of leukocytes adhering to postcapillary venules in the microcirculation (Fig 2, B). The number of adherent leukocytes at 180 minutes after endotoxemia was elevated (P < .003 vs SHAM group) and remained high in the CON and LAV groups. However, in animals receiving FOY lavage, their number decreased already at 240 minutes and
was reduced significantly at 300 minutes after endotoxin administration (P < .001 compared with the CON and LAV groups). The increased leukocyte membrane adhesion in CON and LAV animals was also detected by a decreased LAI at 300 minutes after endotoxin administration (Fig 2, C) (P < .01 vs values at 180 minutes). In the FOY group, the effect was attenuated at 300 minutes after endotoxin application (P < .008 vs CON and LAV group). In some arterioles, rolling and adherent leukocytes were observed in CON and LAV rats at 240 and 300 minutes after endotoxemia (Table I). We did not observe rolling or adherent leukocytes in arterioles of FOY animals. Microvascular cell death and capillary occlusion. Cell death in the cremaster tissue was almost undetectable until 4 hours after endotoxin administration (Fig 2, D). But in the CON and LAV
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Table I. Microvascular parameters in cremaster muscle at different times after endotoxin administration (4 mg/kg IV) Functional capillary density Cap/field SHAM CON LAV FOY
180 min 0.86 0.69 0.45 0.47
± ± ± ±
240 min
0.05 0.24 0.24 0.30
0.65 0.51 0.46 0.62
± ± ± ±
0.11 0.33 0.28 0.31
300 min 0.71 0.31 0.25 0.62
± ± ± ±
0.16 0.19*y 0.09* 0.23
Rolling leukocytes in arterioles Cells/min SHAM CON LAV FOY
180 min 0 4 0 0
± ± ± ±
0 9 0 0
240 min
300 min
0 13 9 0
0 11 19 0
± ± ± ±
0 16 10 0
± ± ± ±
0 12 26 0
Adherent leukocytes in arterioles Cells/mm2 SHAM CON LAV FOY
180 min 0 4 0 0
± ± ± ±
0 8 0 0
240 min 0 4 2 0
± ± ± ±
0 6 2 0
300 min 0 7 4 0
± ± ± ±
0 10 5 0
Functional capillary density = the fraction of not-perfused capillaries in the cremaster muscle in 3 different tissue regions (area about 400 lm 3 400 lm). The number of rolling leukocytes in precapillary arterioles passing a given observation point and counted over 30 seconds (cells/min). The number of adherent leukocytes (without movement for at least 30 seconds) per vessel area measured in an arteriolar vessel segment length of 100 lm (cells/mm2). Mean ± SD are shown. *P < .05 compared with SHAM and FOY at the same time point. yP < .05 compared with value at 180 minutes in the same group.
groups, cell death was increased at 300 minutes compared with the respective values at 180 minutes (P < .001) or the values in the SHAM group (P < .001). The majority of the propidium iodidepositive cells were in the extravascular tissue. At 300 minutes after endotoxin administration, cell death in the cremaster muscle tissue of the FOY group was reduced compared with the CON and LAV groups (P < .001) (Fig 2, D). Functional capillary density in the cremaster muscle was reduced in the CON and LAV groups at 300 minutes after endotoxin administration when compared with the SHAM rats (P < .001) (Table I). At 300 minutes, the FOY group had an improved capillary perfusion with greater average functional capillary density than in the CON and LAV groups (P < .001). IL-6 plasma levels. IL-6 levels at 300 minutes after endotoxemia were elevated significantly in CON and LAV rats compared with their respective baseline values before endotoxin administration (Fig 3). In the FOY group, this effect was attenuated (P < .009 vs CON and LAV groups). The FOY group, however, still showed a significant increase in the IL-6 levels at 300 minutes when compared with the corresponding values before endotoxemia.
Leukocyte activation. The intestinal homogenates of CON and LAV animals 300 minutes after endotoxin administration activated naive human leukocytes (P < .002 vs SHAM) (Fig 4, A). A similar pattern was observed with plasma of the CON and LAV groups (P < .001 vs SHAM) (Fig 4, B). In the FOY group, the level of leukocyte activation produced by the intestinal homogenates (Fig 4, A) and by the plasma (Fig 4, B) were attenuated (P < .002 vs the CON and LAV groups), but remained higher than in the SHAM group. Intestinal homogenates from SHAM animals, which were prepared after rinsing the intestine with either Krebs-Henseleit buffer or with FOY, did not activate human leukocytes (4 ± 2 activated leukocytes/100 cells and 4 ± 1 activated leukocytes/100 cells). FOY alone did not activate human leukocytes (3 ± 2 activated leukocytes/100 cells). After intestinal lavage with either KrebsHenseleit buffer or with FOY, the lavage fluid did not activate human leukocytes (15 ± 12 activated leukocytes/100 cells and 18 ± 6 activated leukocytes/100 cells). Intestinal tissue structure. In the LAV and CON groups, endotoxin administration resulted in comparable intestinal tissue damage with vacuolated
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Fig 3. Plasma interleukin-6 levels in femoral arterial blood measured 300 minutes after intravenous endotoxin administration (4 mg/kg). Groups are the same as in Fig 1. *P < .05 compared with baseline values before endotoxin application. yP < .05 compared with FOY at the same time point.
epithelial cells at the tip of the villi, extrusion/ sloughing of the epithelial cells, mild edema in the lamina propria, and some loss of adhesion between the epithelial cells. We also observed loss of the brush border cells in those rats with early formation of villus digestion after endotoxemia. Damage in the CON and LAV groups was noted only in the upper half of the villi, and no damage was observed from the crypts to the serosa. The FOY group seemed to exhibit less intestinal damage when compared with the LAV and CON group. The FOY rats had only mild extrusion of epithelial cells (Fig 5). IV administration of FOY versus IPI. The FOYiv group exhibited no attenuation of tachycardia and hyperventilation at 300 minutes after endotoxin administration (Table II). Only the blood pressure was slightly improved compared with the CON group but remained significantly reduced compared with SHAM animals. Compared with the FOY group, the FOYiv group had a higher respiratory rate at 300 minutes and higher heart rate at 240 and 300 minutes after endotoxemia (P < .003). DISCUSSION Although the possibility that pancreatic proteases may play a role in shock has been mentioned in the past,30 no evidence for or against this hypothesis has been advanced. There were suggestions that proteases may be involved in shock-induced mucosal injury.31,32 Lefer and his collaborators observed a relationship between pancreatic proteases and a myocardial depressant activity,33 suggesting that intestinal proteases may
Fig 4. Neutrophil activation measured by pseudopod formation of naive human leukocytes mixed with (A) intestine homogenates, and (B) plasma before and after 300 minutes of intravenous endotoxin administration into rats. The increase in activating factors at 240 and 300 minutes after endotoxin administration was significantly reduced by intraintestinal pancreatic protease inhibition with FOY. *P < .05 compared with SHAM. yP < .05 compared with FOY at the same time point.
be involved in the development of cardiac insufficiency during shock. Recently, we obtained direct evidence that pancreatic homogenates alone as well as several abdominal organ homogenates mixed with trypsin or chymotrypsin—but not without them—generate potent inflammatory mediators, which, among several pathophysiologic activities, also cause leukocyte activation. The generation of the inflammatory mediator from these organ homogenates can be attenuated by nonspecific protease inhibition.21 The pancreatic homogenates exhibit myocardial depressor activity.20 Intravenous administration of a potent protease inhibitor did not restore blood pressure to normal levels during shock induced by
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Table II. Cardiopulmonary parameters before (baseline) and after IV endotoxin injection Mean arterial blood pressure (mm Hg) Experimental groups
Baseline
240 min
300 min
SHAM CON FOYiv
110 ± 12 114 ± 12 107 ± 6
105 ± 9 91 ± 11* 109 ± 4
105 ± 8 81 ± 12*y 99 ± 9
Heart rate (beats/min) SHAM CON FOYiv
376 ± 12 398 ± 11 391 ± 8
396 ± 9 491 ± 52*y 413 ± 12*y
377 ± 16 490 ± 69*y 413 ± 12*y
Respiratory rate (breaths/min) SHAM CON FOYiv
73 ± 18 76 ± 17 65 ± 5
70 ± 10 96 ± 15*y 103 ± 6*y
66 ± 10 101 ± 17*y 91 ± 6*y
Sham and CON are the same as in Figure 1. FOYiv (n = 5). This group received endotoxin (4 mg/kg over 5 minutes) and gabexate mesilate (FOY7) intravenously during 5 hours every 30 minutes at a rate of 10 mg/kg/h. This dosage has been shown to inhibit leukocyte activation and cytokine expression during ischemia and reperfusion and shock. In our experiment this dosage given intravenously had no significant improving effect on hemodynamics (except blood pressure) during endotoxemia, whereas intraintestinal protease inhibition with FOY7 significantly improved hemodynamics. Data are presented as mean ± SD. *Significantly different when compared with baseline value on the same group. ySignificantly different when compared with SHAM.
Fig 5. Representative histology sections of the rat small intestine at 5 hours after endotoxin administration. Groups are the same as in Fig 1. Endotoxemia-induced mucosal damage, loss of the brush border with leukocyte infiltration in control groups with and without intestinal lavage (CON, LAV). Intestinal protease inhibition reduced intestinal damage during endotoxemia (FOY).
splanchnic ischemia. Instead, IPI attenuated shockinduced hypotension by reducing the level of inflammatory mediators in the plasma.18,19 The pathophysiology of splanchnic ischemia, however, differs from endotoxin-induced shock. In septic shock, endotoxin can serve as a direct activating factor. As an activator for leukocytes or endothelial cells, endotoxin may cause lowered perfusion and ischemia. During splanchnic ischemia, the biochemical identity of the main trigger mechanism for leukocyte activation is still unknown. New activators may be formed, quite independent of endotoxin. Thus it is interesting to examine whether IPI may also improve endotoxin-induced shock parameters. In the current model of endotoxemia, IPI did not interfere with hemodynamic parameters during the first 20 to 60 minutes, indicating that at the beginning of endotoxemia, intestinal proteases are
not the main source for hemodynamic alterations. At a later time, however, IPI reduced endotoxininduced hypotension, tachycardia, and hyperventilation, and to microvascular and hemodynamic improvement by attenuation of a delayed inflammatory process after the initial endotoxin administration. Inflammation seems to be a major cause for shock and multiple organ failure during sepsis. Inflammation is accompanied by a powerful form of activation that includes leukocytes, endothelial cells, and possibly other cell types.34 Leukocyte activation leads to pseudopod formation and capillary plugging with leukocyte migration across the endothelial wall, local tissue inflammation, and cell death.11 Inflammation is also accompanied by a series of pathophysiologic processes, which may lead to cardiopulmonary dysfunction, including hypotension, tachycardia, and hyperventilation. The attenuation of inflammation with IPI may be one of the major reasons for the improvement of the microvascular perfusion. The biochemical structure of the inflammatory mediators produced in the intestinal wall is the subject of our current investigations. From pancreatic homogenates we have identified leukocyte-
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activating factors with hydrophilic and hydrophobic characteristics and a range of molecular weights, some of them less than 3kD.4,21 Proteases themselves may be less involved in activation of leukocytes,21 supporting the hypothesis that proteases have more of a role as producers of inflammatory mediators. That IPI inhibits the formation of inflammatory mediators supports the hypothesis that such mediators may be predominantly produced within the wall of the intestine by pancreatic digestive enzymes. IPI also reduced endotoxin-induced microvascular leukocyte and endothelial cell activation, parenchymal cell death, and the production of IL-6. Sepsis is accompanied by intestinal ischemia and mucosal cell injury.35-38 Elevation of the mucosal epithelial permeability permits entry of pancreatic enzymes into the interstitium of the intestinal wall. Entry of enzymes into the submucosal interstitium may lead to generation of inflammatory mediators by proteolytic cleavage within the intestinal wall. Our findings support this hypothesis, because IPI served to reduce the generation of activating factors in the small intestine as well as mucosal damage during endotoxemia. Additional aspects of this hypothesis remain to be examined. It may be argued that intestinal endotoxin or intestine-derived cytokines may be blocked by IPI. Lavage of the intestine was less effective in preventing the systemic inflammation than was lavage combined with protease inhibition. We investigated the relation between IPI, endotoxin activity, and tumor necrosis factor alpha (TNF-a) levels during intestinal ischemia-induced shock (unpublished results). IPI did not reduce intestinal endotoxin activity. The levels of TNF-a in intestinal homogenates during shock were identical in IPI and control animals. Endotoxin activity and TNF-a levels did not correlate with the amount of leukocyte activation either in the intestine or in the plasma. Taken together, this evidence suggests that intestinal endotoxin and intestine-derived TNF-a may be less involved in the mechanisms by which IPI attenuates shock. We hypothesize that intestinal absorption of FOY and inhibition of leukocyte activation and adhesion,39 as well as reduction of cytokine levels,40 may in part account for the improved hemodynamics and decreased microvascular inflammation in the current experiments. However, IV-administered FOY at a dose known to reduce leukocyte activation28 did not improve hemodynamics during sepsis in our model. Only endotoxin-induced hypotension was partially attenuated in this group,
possibly as a result of the inhibitory effect of FOY on the constitutive and inducible nitric oxide synthase.41 Our results do not clarify whether FOY leaking into the extraintestinal tissue may, in part, have accounted for the effects seen in the FOY group. Further studies are required with other protease inhibitors. Intestine-derived oxygen radicals may be influenced by IPI. Recent data demonstrated that IPI did not affect either xanthine dehydrogenase to xanthine oxidase conversion or xanthine oxidase activity in the ischemic intestine. The addition of allopurinol to the lavage fluid during IPI did not increase the beneficial effect of IPI even though allopurinol inhibited the xanthine oxidase activity in the ischemic intestine.19 Systemic leukocyte activation and shock may be related more closely to protease activity than to the action of oxygen free radicals derived from xanthine oxidase. In conclusion, we provide here the first evidence to suggest that, in addition to endotoxin, intestinal proteases may be involved in the generation of intestine-derived inflammatory mediators in the plasma. These mediators may sustain systemic cell activation and produce organ injury after an endotoxin challenge. Intraintestinal pancreatic protease blockade may have clinical utility in septic shock patients. The authors wish to thank Mr William H. Loomis, UCSD Department of Surgery, for technical help with the IL-6 measurements, and Dr Tony Hugli, La Jolla Institute for Molecular Medicine, for his thoughtful suggestions regarding this study. FOYÒ was a gift from Ono Pharmaceutical.
REFERENCES 1. Mazzoni MC, Schmid-Scho¨bein GW. Mechanisms and consequences of cell activation in the microcirculation. Cardiovasc Res 1996;32:709-19. 2. Parillo JE, Burch C, Shelhamer JH, Parker MM, Natanson C, Schuette W. A circulating myocardial depressing substance in humans with septic shock. J Clin Invest 1985;76:1539-53. 3. Haglund U. Systemic mediators released from the gut in critical illness. Crit Care Med 1993;21:S15-S18. 4. Schmid-Scho¨nbein GW, Kistler EK, Hugli TE. Mechanisms for cell activation and its consequences for biorheology and microcirculation: Multiple-organ failure in shock. Biorheol 2001;38:185-201. 5. Yang S, Koo DJ, Chaudry IH, Wang P. The important role of the gut in initiating the hyperdynamic response during early sepsis. J Surg Res 2000;89:31-7. 6. Evans WE, Darin JC. Effect of enterectomy in endotoxin shock. Surgery 1966;60:1026-9. 7. Hassoun HT, Kone BC, Mercer DW, Moody FG, Weisbrodt HW, Moore FA. Post-injury multiple organ failure: the role of the gut. Shock 2001;15:1-10.
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8. Tamion F, Richard V, Lyoumi S, et al. Gut ischemia and mesenteric synthesis of inflammatory cytokines after hemorrhagic or endotoxic shock. Am J Physiol 1997;273: G314-21. 9. Adams CA, Xu DZ, Deitch EA. Factors larger than 100kD in posthemorrhagic shock mesenteric lymph are toxic for endothelial cells. Surgery 2001;129:351-62. 10. Deitch EA, Adams C, Lu Q, Xu DZ. A time course study of the protective effect of mesenteric lymph duct ligation on hemorrhagic shock-induced pulmonary injury and the toxic effects of lymph from shocked rats on endothelial cell monolayer permeability. Surgery 2001;129:39-47. 11. Schmid-Scho¨nbein GW, Hugli TE, Kistler EB, Sofianos A, Mitsuoka H. Pancreatic enzymes and microvascular cell activation in multiorgan failure. Microcirc 2001;8:5-14. 12. Hardaway R. A review of septic shock. Am Surg 2000;66: 22-9. 13. Zeni F, Freeman B, Natanson C. Anti-inflammatory therapies to treat sepsis and septic shock. A reassessment. Crit Care Med 1997;25:1095-100. 14. Mainous MR, Ertel W, Chaudry IH, Deitch EA. The gut: a cytokine generating organ in systemic inflammation? Shock 1995;4:193-9. 15. Deitch EA, Xu D, Franko L, Ayala A, Chaudry IH. Evidence favoring the role of the gut as a cytokine-generating organ in rats subjected to hemorrhagic shock. Shock 1994;1:141-6. 16. Koike K, Moore EE, Moore FA, Read RA, Carl VS, Banerjee A. Gut ischemia/reperfusion produces lung injury independent of endotoxin. Crit Care Med 1994;22:1438-44. 17. Bone RC. Why sepsis trials fail. JAMA 1996;276:565-6. 18. Mitsuoka H, Kistler EB, Schmid-Scho¨nbein GW. Generation of in vivo activating factors in the ischemic intestine by pancreatic enzymes. PNAS 2000;97:1772-7. 19. Mitsuoka H, Kistler EB, Schmid-Scho¨nbein GW. Protease inhibition in the intestinal lumen: Attenuation of inflammation and early indicators of multiple organ failure in shock. Shock 2002;17:205-9. 20. Kistler EB, Lefer AM, Hugli TE, Schmid-Scho¨nbein GW. Plasma activation during splanchnic arterial occlusion shock. Shock 2000;14:30-4. 21. Kistler EB, Hugli TE, Schmid-Scho¨nbein GW. The pancreas as a source of cardiovascular cell activating factors. Microcirc 2000;7:183-92. 22. Mitsuoka H, Schmid-Scho¨nbein GW. Mechanisms for blockade of in vivo activator production in the ischemic intestine and multi-organ failure. Shock 2000;14:522-7. 23. Fitzal F, De Lano F, Young C, Rosario HS, SchmidScho¨nbein GW. Intestinal pancreatic protease inhibition attenuates intestinal-ischemia-induced shock by reducing cell activation and gut injury. J Vasc Res 2002;39:320-9. 24. Baez S. An open cremaster muscle preparation for the study of blood vessels by in vivo microscopy. Microvas Res 1973;5:384-94. 25. Suematsu M, DeLano FA, Poole D, Engler RL, Miyasaka M, Zweifach BW, Schmid-Scho¨nbein GW. Spatial and temporal
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correlation between leukocyte behavior and cell injury in postischemic rat skeletal muscle. Lab Invest 1994;70:684-95. Schmid-Scho¨nbein GW, Skalak R, Simon SI, Engler RL. The interaction between leukocytes and endothelium in-vivo. Proc NY Acad Sci 1988;516:348-61. Aosasa S, Ono S, Mochizzuki H, Tsujimoto H, Ueno C, Matsumoto A. Mechanism of the inhibitory effect of protease inhibitor on tumor necrosis factor alpha production of monocytes. Shock 2001;15:101-5. Harada N, Okajima K, Mushimoto S. Gabexate mesylate, a synthetic protease inhibitor, reduces ischemia/reperfusion injury of rat liver by inhibiting leukocyte activation. Crit Care Med 1999;27:1958-64. Ohashi I, Nishijima J, Murata A, et al. Inhibitory effect of a synthetic protease inhibitor (gabexate mesylate) in the respiratory burst oxidase in human neutrophils. J Biochem 1995;118:1001-6. Bounous G. Mucosal injury in low flow states. Can J Surg 1974;17:434. Montgomery A, Borgstro¨m A, Haglund U. Pancreatic proteases and intestinal mucosal injury after ischemia and reperfusion in the pig. Gastronterology 1992;102:216-22. Bounous G. Pancreatic proteases and oxygen-derived free radicals in acute ischemic enteropathy. Surgery 1985;99: 92-4. Lefer AM, Spath JA. Pancreatic hypoperfusion and the production of a myocardial depressant factor in hemorrhagic shock. Ann Surg 1974;179:868-76. Nieuwenhuijzen GAP, Haskel Y, Lu Q, et al. Macrophage elimination increases bacterial translocation and gut-originsepticemia, but attenuates symptoms and mortality rate in a model of systemic inflammation. Ann Surg 1993;218: 791-9. Turnbull RG, Talbot JA, Hamilton SM. Hemodynamic changes in gut barrier function in sequential hemorrhagic and endotoxic shock. Crit Care Med 1995;38:705-12. Oldner A, Goiny M, Rudehill A, Ungerstedt U, Sollevi A. Tissue hypoxanthine reflects gut vulnerability in porcine endotoxin shock. Crit Care Med 1999;27:790-7. Smithies M, Yee TH, Jackson L, Beale R, Bihari D. Protecting the gut and the liver in the critically ill: effects of dopexamine. Crit Care Med 1994;22:789-95. McCarthy Pastores S, Katz DP, Kvetan V. Splanchnic ischemia and gut mucosal injury in sepsis and the multiple organ dysfunction syndrome. Am J Gastroenterol 1996;91: 1697-710. Lu SP, Tsai CC, Shau WY, et al. Effects of gabexate mesilate (FOY) on ischemia-reperfusion-induced acute lung injury in dogs. J Surg Res 1999;87:152-63. Malyon AD, Husein M, Weiler-Mithoff EM. How many procedures to make a breast? Br J Plast Surg 2001;54: 227-31. Colasanti M, Persichini T, Venturini G, Menegatti E, Lauro GM, Ascenzi P. Effect of gabexate mesylate (FOY), a drug for serine proteinase-mediated diseases on the nitric oxide pathway. Biochem Biophys Res Commun 1998;246:453-6.
Background. Sepsis is accompanied by severe inflammation whose mechanism remains uncertain. We recently demonstrated that pancreatic proteases in the ischemic intestine have the ability to generate powerful inflammatory mediators that can be detected in the portal vein and in the general circulation. This study was designed to examine several circulatory and inflammatory indices during experimental endotoxemia and intraintestinal pancreatic protease inhibition. Methods. Immediately after intravenous endotoxin administration, the small intestine was subjected to intraluminal lavage with and without gabexate mesilate, an inhibitor of pancreatic proteases. Shams and rats without lavage served as controls. Hemodynamics, leukocyte (neutrophil and monocyte), and endothelial cell activation, as well as organ injury in the intestine and the cremaster muscle, were examined. Results. After endotoxin administration, control rats developed hypotension, tachycardia, hyperventilation, and leukopenia. The intestine and plasma contained mediators that activated leukocytes. The leukocyte-endothelial interaction within the cremaster muscle microcirculation was enhanced. Endotoxin administration resulted in elevated interleukin-6 plasma levels. Histologic evidence indicated liver and intestinal injury. In contrast, blockade of pancreatic proteases in the intestinal lumen significantly improved hemodynamic parameters and reduced all indices of inflammation in plasma and cell injury in skeletal muscle microcirculation. Conclusions. Inflammatory mediators derived from the intestine by pancreatic proteases may be involved in the prolonged inflammatory response and sustain symptoms of sepsis after endotoxin challenge. (Surgery 2003;134:446-56.) From the Department of Bioengineering, the Whitaker Institute for Biomedical Engineering and Department of Surgery, UCSD Medical Center, University of California–San Diego, La Jolla, Calif; Department of Surgery, University of Vienna Medical School, and Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria; and Institute of Biochemistry, Faculty of Medicine of Lisbon, Lisbon, Portugal
SYSTEMIC INFLAMMATION (also denoted as systemic inflammatory response syndrome) and sepsis remain the leading causes of death in intensive care units. Evidence indicates that the severity of inflammation as measured by leukocyte (neutrophil and monocyte) activation in the circulation may serve as a predictor for survival,1 and that inThis work was supported by the Max Kade Foundation, and in part by NIH Grants HL-43026, HL-67825, GM-60475, and by FLAD 460/2000. Accepted for publication April 12, 2003. Reprint requests: Florian Fitzal, MD, Department of Surgery, University of Vienna Medical School, Waehringer Guertel 18-20, 1090 Vienna, Austria. Ó 2003, Mosby, Inc. All rights reserved. 0039-6060/2003/$30.00 + 0 doi:10.1067/S0039-6060(03)00168-5
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flammatory mediators are produced during sepsis. These inflammatory mediators are detectable in plasma and exhibit a variety of activities which, in addition to the activation of circulating leukocytes, include suppression of T lymphocyte proliferation and myocardial contraction, as well as several other cell and organ dysfunctions.2-4 The intestine appears to play a central role during sepsis.5,6 It generates different inflammatory mediators,7-10 some of which have been characterized (tumor necrosis factor, interleukins, leukotrienes, platelet activating factor), and some of which remain unidentified.9,11 Translocation of intestinal bacteria and endotoxin has been suggested to be responsible for the prolonged inflammation during sepsis. However, a number of attempts to relate intestinal-derived endotoxemia to mortality rates in clinical sepsis
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studies have failed so far.12,13 The intestine may generate factors that activate inflammatory cells even in the absence of endotoxin or bacteria.14-16 Thus, we may have to reexamine our basic hypothesis for the pathophysiology of sepsis because other inflammatory mediators besides endotoxin, cytokines, or lipid-derived mediators may produce cell activation and multiple organ injury.12,17 Recently we demonstrated an important role of pancreatic digestive enzymes during multiorgan failure following intestinal ischemia. After entry into the intestinal tissue because of ischemiainduced breakdown of the mucosal barrier, digestive enzymes may generate a set of powerful inflammatory mediators in the intestinal tissue that activate leukocytes and endothelial cells in the microcirculation.18,19 The mediators produced by the digestive enzymes enhance the oxidative stress, cause myocardial depression, and lead to apoptotic cell death in the tissue parenchyma and rapid mortality.11,20,21 Inhibition of pancreatic proteases in the lumen of the intestine significantly improves the hemodynamics and reduces inflammation during intestinal ischemia/reperfusion-induced shock.19,22,23 In endotoxin shock, however, the impact of the inflammatory mediators derived from pancreatic proteases on the cardiovascular system is unexplored. We hypothesize that sepsis-induced hypoperfusion of the intestine and elevated permeability of the mucosal epithelium may lead to translocation of pancreatic enzymes from the lumen into the wall of the intestine, followed by generation of factors that activate inflammatory cells within the wall. The inflammatory mediators may then enter the circulation and maintain and enhance inflammation during sepsis, even though it may have been triggered by endotoxin. We examine here one particular aspect of this hypothesis. After endotoxin administration, we study the effect of intraintestinal pancreatic protease inhibition (IPI) on central cardiopulmonary dynamics, systemic inflammation, the level of inflammatory mediators that cause leukocyte activation, and peripheral muscle parenchymal cell death. MATERIAL AND METHODS Twenty-five male Wistar rats (180-220 g; Charles River Breeding Laboratories, Wilmington, Mass) were maintained on a standard rat chow and water ad libitum. All experiments were reviewed and approved by the University of California San Diego Animal Subjects Committee. The rats were fasted overnight. After general anesthesia (60 mg/kg
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intramuscular pentobarbital), the left carotid artery and right jugular vein were cannulated (polyethylene tubing, PE-50, Clay Adams, Parsippany, NJ) to record arterial pressure and heart rate and to administer supplemental anesthetic agent or fluid support (Ringer’s lactate 2 mL/h), respectively. A tracheal tube was inserted to minimize respiratory complications. Escherichia coli-derived endotoxin (serotype 127:B8, Sigma Chemical Co, St. Louis, Mo; 4 mg/kg) was administered intravenously into the jugular vein over 5 min. Each animal was then observed during the subsequent 5-hour period. Experimental groups. The rats were divided randomly into 4 groups (each with 5 rats): 1. SHAM group: sham surgery without endotoxin administration or intestinal lavage 2. CON group: endotoxin administration without intestinal lavage 3. LAV group: endotoxin administration and intestinal lavage with 450 mL of Krebs-Henseleit buffer (see the following sections) 4. FOY group: endotoxin administration and intestinal lavage with 450 mL of Krebs-Henseleit buffer with 0.38 mmol/L of the protease inhibitor gabexate mesilate (FOY; Ono Pharmaceutical, Tokyo, Japan). Intestinal lavage and IPI. In the LAV and FOY groups, a polyethylene tube (PE 250, Clay Adams) was inserted into the duodenal lumen immediately distal to the pylorus (fixed with silk 4/0) and another tube was placed into the terminal ileum to permit free fluid discharge into a collection container. At the onset of endotoxin administration, the small intestinal lumen was rinsed gently (10 mL/min) with 100 mL of Krebs-Henseleit buffer solution (7.7 g NaCl, 0.35 g KCl, 0.29 g CaCl2, 0.3 g MgSO4 per liter of sterile water, 388C, pH 7.6-7.8) without (LAV group) or with FOY for intestinal protease blockade (FOY group). Also in the LAV and FOY groups, the small intestinal lumen was rinsed gently (10 mL/min) every 30 minutes with another 50 mL of buffer (with or without FOY, respectively). Pilot studies with intravital microscopy showed that intestinal lavage did not alter the intestinal microcirculation, either with or without FOY (unpublished data). Blood pressure dropped transiently in some rats during the first 5 minutes of lavage, leading to a short period (about 1 minute) of hypotension, with blood pressures of 60 mm Hg. We did not observe an increase in blood pressure resulting from intestinal lavage. In preliminary studies we examined endotoxin activity by the limulus
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amebocyte lysate activity test (unpublished results) in the intestinal fluid before and after intestinal lavage (n = 5 per group). Intestinal lavage with or without FOY did not influence endotoxin activity in the intestinal fluid when compared with prelavage levels (LAV group, prelavage mean ± SD: 140 ± 179 vs postlavage 86 ± 36 EU/mL endotoxin activity; FOY group, prelavage mean ± SD: 66 ± 72 vs postlavage 41 ± 20 EU/mL endotoxin activity). Cremaster muscle microcirculation. To examine leukocyte and endothelial cell activation in the peripheral microcirculation, the cremaster muscle was exposed for intravital microscopy24 at 120 minutes after endotoxin administration. The muscle preparation was continuously superfused with Krebs-Henseleit bicarbonate-buffered solution saturated with a mixture of 95% N2 and 5% CO2 at 378C. In each muscle, 1 terminal arteriole (30-50 lm diameter), 3 capillary observation fields (20 to 30 capillaries/field), and 3 postcapillary venules (2030 lm diameter) in the central portion of the muscle were examined with a 320 objective (numeric aperture = 0.5) for the capillary fields and the arterioles and venules with a 340 water-immersion objective (numeric aperture = 0.76). After a 30-minute adaptation period, the first microvascular bright field and fluorescent images were recorded with a color CCD camera (model VI470, Optronics, Goleta, Calif) and a videotape recorder (AG6300, Panasonic; Matsushita Electric Ind Co, Japan) for offline data analysis. Propidium iodide (Sigma) was added to the cremaster muscle superfusate (1 lmol/L final concentration) to label the nuclei of dead cells.25 At each time point, the images of the microvessels were recorded for 1 minute; otherwise, the tissue was not illuminated to minimize tissue injury because of the exposure of fluorescent light. Images of the muscle microcirculation were recorded 180, 240, and 300 minutes after endotoxin administration. Microvascular measurements. Red blood cell velocity (VRBC) was measured online by the crosscorrelation technique (Vista Electronics Company, Ramona, Calif). Vessel diameters and lengths as well as the number of rolling and adherent leukocytes in the pre-capillary arterioles and in postcapillary venules at each time point were determined. Leukocytes that were rolling continuously along the vessel wall were counted per unit time. Leukocytes attached firmly to the endothelium for a period of 30 seconds or longer were referred to as adherent cells. Leukocyte rolling velocity (VWBC) on the endothelium in postcapillary venules was measured off-
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line from the displacement of individual leukocytes from image frame to frame (time duration for 1 video frame = 1/30 sec). The leukocyte adhesion index (LAI) was calculated from the ratio of VWBC and VRBC in the same postcapillary venule segment measured during the same time interval. LAI falls between 0 and 1. A decline in LAI results from an increase in leukocyte-endothelial cell membrane attachment.26 In each microvascular field, functional capillary density was determined as the fraction of perfused capillaries and the number of all (perfused and nonperfused) capillaries. Tissue samples. Before intestinal lavage and at the end of the experiment, intestinal fluid was collected from the ileal tube and centrifuged at 500 g for 5 minutes. Aliquots were stored (708C) until use for measurement of protease activity (see the following section). One aliquot of blood (1 mL containing 10 U/mL heparin) was collected before endotoxin administration, and 1 aliquot at the end of the experiment. The samples were centrifuged (500 g for 5 minutes) and the plasma stored (708C) for measurement of leukocyte activation and IL-6 levels (see the following section). An additional 0.1 mL of heparinized blood was used to determine the leukocyte counts in peripheral blood (Unopette Microcollection System, Bekton, NJ). After euthanasia (5 hours after endotoxin administration), a segment of the ileum (5 cm) was excised, rinsed in phosphate buffered saline (PBS, 10 mmol/L) and fixed in 3% formalin (24 hours). The intestinal segments were embedded in resin, sectioned (1 lm section thickness), stained with toluidine blue, and assessed by light microscopy for morphologic damage (no quantification, 3 sections per animal). Another segment of the terminal ileum (1 g) was excised, and all intestinal contents were rinsed. The segment was placed into 3 mL of a PBS solution, homogenized, and centrifuged at 1000 g for 10 minutes. The supernatants from the homogenate were collected and stored (708C) for later measurements of leukocyte activation as pseudopod formation (see the following section). Protease activity assay. Total protease activity in intestinal fluid was measured with a protease assay kit (EnzChek, Molecular Probes, Eugene, Ore). The protease activities detected by this kit included elastase, trypsin, chymotrypsin and pepsin. The fluorescence intensity of the kit mixed with the samples was measured with a fluorometer and enzyme activity was expressed in fluorescence units (FU) with a lower detection limit between 1.0 3 103 and 4.4 3 105 FU.
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Leukocyte activation. Supernatant samples of the intestinal homogenates and plasma samples were mixed with naive human leukocytes (from asymptomatic volunteers). The inflammatory tripeptide F-Met-Leu-Phe (107 mol/L) and buffer served as positive and negative controls, respectively (not shown). Exposure of the leukocytes leads to general activation (eg, superoxide formation, degranulation, expression of adhesion molecules, polymerization of actin with pseudopod formation, and mechanical stiffening of the cytoplasm). Pseudopod formation leads to leukocyte entrapment in the microcirculation and migration into extravascular tissue. Thus, as an index of leukocyte activation, we determined the fraction of cells with pseudopod formation as described earlier.21 One hundred leukocytes (neutrophils and monocytes) were microscopically examined (1003 objective, numerical aperture 1.4, 103 objective) and cells with cytoplasmic projections greater than 1 lm were classified as activated leukocytes. IL-6 measurements. IL-6 levels of prediluted plasma samples (1:100) were measured with a commercial ELISA kit (Quantikine M ELISA; R&D Systems Inc., Minneapolis, Minn). Samples were measured in duplicates and values were expressed as ng/mL. IV administration of gabexate mesylate. During intestinal lavage, FOY may be partially absorbed into the circulation, where it can reduce cytokine levels,27 leukocyte activation,28 and oxygen-free radical formation,29 and therefore may influence parameters of septic shock. To investigate whether intestinal lavage with FOY affects the hemodynamics by way of intestinal protease inhibition or by direct intravascular systemic actions, 5 rats were prepared as described previously. Endotoxin administration (4 mg/kg) was followed by 8 FOY bolus administrations (10 mg/kg/h) into the jugular vein every 30 minutes (FOYiv group). This dose is sufficient to reduce leukocyte adhesion and attenuate ischemia/reperfusion injury.28 Hemodynamic parameters were measured as described previously. Statistical analysis. Data are presented as mean ± standard deviation. Repeated measurements of protease activity in intestinal fluids and the plasma levels of interleukin (IL)-6 were analyzed using the paired Student t test. The Mann-Whitney Rank Sum test was used to assess differences in trypsin activity between the 2 groups. Repeated measurements of microvascular and hemodynamic parameters in the same group were compared with repeated analysis of variance (ANOVA). For normally distributed data, ANOVA was followed by the Tukey’s multiple comparison correction test (in comparison with
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control baseline); for Poisson distributed data, the ANOVA on ranks was followed by Dunnett’s correction test. ANOVA was used to assess differences between groups for microcirculatory, hemodynamic, and IL-6 measurements as well as leukocyte activation at the same time. ANOVA was followed by Tukey’s correction test for normally distributed data, for Poisson distributed data, and ANOVA on ranks was followed by Student Newman Keuls’ correction test; P < .05 was considered to be significant with a power of .8. RESULTS Intraintestinal pancreatic protease inhibition (IPI). Intraintestinal lavage with FOY served to reduce intestinal serine protease activity (FOY group, 92 ± 17 units) compared with its baseline value (CON group, 2604 ± 734 units, P < .006) or with intestinal protease activity after lavage with vehicle (LAV group, 630 ± 441 units, P < .029). Hemodynamic parameters. Endotoxin administration in the CON and LAV groups was followed by a significant reduction in mean arterial blood pressure compared with the baseline and with the SHAM group. In contrast, after an initial drop at 20 minutes, the arterial blood pressure in the FOY group increased to pre-endotoxemia levels and remained stable thereafter. Throughout the rest of the experiment there were no significant differences in femoral pressure between SHAM, FOY, and their respective baseline levels. There were also no significant differences in pressure between the FOY and LAV groups (Fig 1, A). The FOY group exhibited a trend (not significant) toward higher arterial pressure than did the LAV group. After endotoxin administration, the heart rate (Fig 1, B) and respiratory rate (Fig 1, C) increased significantly in the CON, LAV, and FOY groups at 60 minutes when compared with the SHAM group. At 180 minutes after endotoxemia, the heart and respiratory rate of the LAV and CON group remained elevated significantly when compared with SHAMs. The FOY group had lower values than the CON group (P < .004), which were not significantly different from the SHAM values. At 300 minutes, the FOY group had lower heart and respiratory rates than the LAV and CON groups (P < .002). Leukocyte counts. Endotoxemia induced a significant leukopenia in venous blood of all groups at 300 minutes (CON: 2410 ± 650 cells/mm3; LAV: 1380 ± 620 cells/mm3; FOY: 3010 ± 2390 cells/ mm3) compared with the SHAM group (7400 ± 1610 cells/mm3; P < .001).
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Fig 1. Cardiopulmonary parameters in rats measured during 300 minutes after endotoxin administration (4 mg/kg intravenously). The groups are: SHAM, CON (controls with endotoxin administration), LAV (endotoxin administration and intestinal lavage with buffer), FOY (endotoxin administration and intestinal lavage with the protease inhibitor FOY, 0.37 mM). Hypotension (A), tachycardia (B), and hyperventilation (C) were attenuated by intraintestinal protease inhibition. Significant differences at P < .05 between LAV compared with its baseline (*); FOY compared with its baseline (y); CON compared with its baseline and SHAM (à); CON compared with its baseline (§); CON, LAV, FOY compared with SHAM (k); LAV compared with FOY (P < .002) (P); FOY compared with CON (P < .004) (#); and LAV compared with SHAM (P < .004) (**); n = 5 rats in each group.
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Fig 2. Systemic cell activation. Leukocyte and endothelial cell activation in the cremaster muscle in postcapillary venules measured by intravital microscopy at 180, 240, and 300 minutes after endotoxin administration. Groups are the same as in Fig 1. A, Number of rolling leukocytes. B, Number of adherent leukocytes to endothelial cells. C, Leukocyte adhesion index (LAI = VWBC/VRBC) as indicator for endothelial/leukocyte activation and interaction. D, The number of propidium iodide (PI) positive parenchymal cremaster cells as marker for peripheral cell death. Data are presented as mean ± SD. *P < .05 compared with SHAM at the same time point. yP < .05 FOY compared with CON and LAV at the same time point. àP < .05 when compared with 180 minutes after endotoxemia.
Microvascular leukocyte kinetics. The number of rolling leukocytes (Fig 2, A) in postcapillary venules of the CON and LAV groups at 240 minutes and 300 minutes (P < .004) after endotoxin administration were increased compared with the SHAM group. In the FOY group, the number of rolling leukocytes did not differ from that of the SHAMs. FOY attenuated the reduction of rolling leukocytes in the CON and LAV groups at 240 and 300 minutes after endotoxemia (P < .004). In the CON and LAV groups, the central leukopenia (see previous) was accompanied by an increased number of leukocytes adhering to postcapillary venules in the microcirculation (Fig 2, B). The number of adherent leukocytes at 180 minutes after endotoxemia was elevated (P < .003 vs SHAM group) and remained high in the CON and LAV groups. However, in animals receiving FOY lavage, their number decreased already at 240 minutes and
was reduced significantly at 300 minutes after endotoxin administration (P < .001 compared with the CON and LAV groups). The increased leukocyte membrane adhesion in CON and LAV animals was also detected by a decreased LAI at 300 minutes after endotoxin administration (Fig 2, C) (P < .01 vs values at 180 minutes). In the FOY group, the effect was attenuated at 300 minutes after endotoxin application (P < .008 vs CON and LAV group). In some arterioles, rolling and adherent leukocytes were observed in CON and LAV rats at 240 and 300 minutes after endotoxemia (Table I). We did not observe rolling or adherent leukocytes in arterioles of FOY animals. Microvascular cell death and capillary occlusion. Cell death in the cremaster tissue was almost undetectable until 4 hours after endotoxin administration (Fig 2, D). But in the CON and LAV
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Table I. Microvascular parameters in cremaster muscle at different times after endotoxin administration (4 mg/kg IV) Functional capillary density Cap/field SHAM CON LAV FOY
180 min 0.86 0.69 0.45 0.47
± ± ± ±
240 min
0.05 0.24 0.24 0.30
0.65 0.51 0.46 0.62
± ± ± ±
0.11 0.33 0.28 0.31
300 min 0.71 0.31 0.25 0.62
± ± ± ±
0.16 0.19*y 0.09* 0.23
Rolling leukocytes in arterioles Cells/min SHAM CON LAV FOY
180 min 0 4 0 0
± ± ± ±
0 9 0 0
240 min
300 min
0 13 9 0
0 11 19 0
± ± ± ±
0 16 10 0
± ± ± ±
0 12 26 0
Adherent leukocytes in arterioles Cells/mm2 SHAM CON LAV FOY
180 min 0 4 0 0
± ± ± ±
0 8 0 0
240 min 0 4 2 0
± ± ± ±
0 6 2 0
300 min 0 7 4 0
± ± ± ±
0 10 5 0
Functional capillary density = the fraction of not-perfused capillaries in the cremaster muscle in 3 different tissue regions (area about 400 lm 3 400 lm). The number of rolling leukocytes in precapillary arterioles passing a given observation point and counted over 30 seconds (cells/min). The number of adherent leukocytes (without movement for at least 30 seconds) per vessel area measured in an arteriolar vessel segment length of 100 lm (cells/mm2). Mean ± SD are shown. *P < .05 compared with SHAM and FOY at the same time point. yP < .05 compared with value at 180 minutes in the same group.
groups, cell death was increased at 300 minutes compared with the respective values at 180 minutes (P < .001) or the values in the SHAM group (P < .001). The majority of the propidium iodidepositive cells were in the extravascular tissue. At 300 minutes after endotoxin administration, cell death in the cremaster muscle tissue of the FOY group was reduced compared with the CON and LAV groups (P < .001) (Fig 2, D). Functional capillary density in the cremaster muscle was reduced in the CON and LAV groups at 300 minutes after endotoxin administration when compared with the SHAM rats (P < .001) (Table I). At 300 minutes, the FOY group had an improved capillary perfusion with greater average functional capillary density than in the CON and LAV groups (P < .001). IL-6 plasma levels. IL-6 levels at 300 minutes after endotoxemia were elevated significantly in CON and LAV rats compared with their respective baseline values before endotoxin administration (Fig 3). In the FOY group, this effect was attenuated (P < .009 vs CON and LAV groups). The FOY group, however, still showed a significant increase in the IL-6 levels at 300 minutes when compared with the corresponding values before endotoxemia.
Leukocyte activation. The intestinal homogenates of CON and LAV animals 300 minutes after endotoxin administration activated naive human leukocytes (P < .002 vs SHAM) (Fig 4, A). A similar pattern was observed with plasma of the CON and LAV groups (P < .001 vs SHAM) (Fig 4, B). In the FOY group, the level of leukocyte activation produced by the intestinal homogenates (Fig 4, A) and by the plasma (Fig 4, B) were attenuated (P < .002 vs the CON and LAV groups), but remained higher than in the SHAM group. Intestinal homogenates from SHAM animals, which were prepared after rinsing the intestine with either Krebs-Henseleit buffer or with FOY, did not activate human leukocytes (4 ± 2 activated leukocytes/100 cells and 4 ± 1 activated leukocytes/100 cells). FOY alone did not activate human leukocytes (3 ± 2 activated leukocytes/100 cells). After intestinal lavage with either KrebsHenseleit buffer or with FOY, the lavage fluid did not activate human leukocytes (15 ± 12 activated leukocytes/100 cells and 18 ± 6 activated leukocytes/100 cells). Intestinal tissue structure. In the LAV and CON groups, endotoxin administration resulted in comparable intestinal tissue damage with vacuolated
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Fig 3. Plasma interleukin-6 levels in femoral arterial blood measured 300 minutes after intravenous endotoxin administration (4 mg/kg). Groups are the same as in Fig 1. *P < .05 compared with baseline values before endotoxin application. yP < .05 compared with FOY at the same time point.
epithelial cells at the tip of the villi, extrusion/ sloughing of the epithelial cells, mild edema in the lamina propria, and some loss of adhesion between the epithelial cells. We also observed loss of the brush border cells in those rats with early formation of villus digestion after endotoxemia. Damage in the CON and LAV groups was noted only in the upper half of the villi, and no damage was observed from the crypts to the serosa. The FOY group seemed to exhibit less intestinal damage when compared with the LAV and CON group. The FOY rats had only mild extrusion of epithelial cells (Fig 5). IV administration of FOY versus IPI. The FOYiv group exhibited no attenuation of tachycardia and hyperventilation at 300 minutes after endotoxin administration (Table II). Only the blood pressure was slightly improved compared with the CON group but remained significantly reduced compared with SHAM animals. Compared with the FOY group, the FOYiv group had a higher respiratory rate at 300 minutes and higher heart rate at 240 and 300 minutes after endotoxemia (P < .003). DISCUSSION Although the possibility that pancreatic proteases may play a role in shock has been mentioned in the past,30 no evidence for or against this hypothesis has been advanced. There were suggestions that proteases may be involved in shock-induced mucosal injury.31,32 Lefer and his collaborators observed a relationship between pancreatic proteases and a myocardial depressant activity,33 suggesting that intestinal proteases may
Fig 4. Neutrophil activation measured by pseudopod formation of naive human leukocytes mixed with (A) intestine homogenates, and (B) plasma before and after 300 minutes of intravenous endotoxin administration into rats. The increase in activating factors at 240 and 300 minutes after endotoxin administration was significantly reduced by intraintestinal pancreatic protease inhibition with FOY. *P < .05 compared with SHAM. yP < .05 compared with FOY at the same time point.
be involved in the development of cardiac insufficiency during shock. Recently, we obtained direct evidence that pancreatic homogenates alone as well as several abdominal organ homogenates mixed with trypsin or chymotrypsin—but not without them—generate potent inflammatory mediators, which, among several pathophysiologic activities, also cause leukocyte activation. The generation of the inflammatory mediator from these organ homogenates can be attenuated by nonspecific protease inhibition.21 The pancreatic homogenates exhibit myocardial depressor activity.20 Intravenous administration of a potent protease inhibitor did not restore blood pressure to normal levels during shock induced by
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Table II. Cardiopulmonary parameters before (baseline) and after IV endotoxin injection Mean arterial blood pressure (mm Hg) Experimental groups
Baseline
240 min
300 min
SHAM CON FOYiv
110 ± 12 114 ± 12 107 ± 6
105 ± 9 91 ± 11* 109 ± 4
105 ± 8 81 ± 12*y 99 ± 9
Heart rate (beats/min) SHAM CON FOYiv
376 ± 12 398 ± 11 391 ± 8
396 ± 9 491 ± 52*y 413 ± 12*y
377 ± 16 490 ± 69*y 413 ± 12*y
Respiratory rate (breaths/min) SHAM CON FOYiv
73 ± 18 76 ± 17 65 ± 5
70 ± 10 96 ± 15*y 103 ± 6*y
66 ± 10 101 ± 17*y 91 ± 6*y
Sham and CON are the same as in Figure 1. FOYiv (n = 5). This group received endotoxin (4 mg/kg over 5 minutes) and gabexate mesilate (FOY7) intravenously during 5 hours every 30 minutes at a rate of 10 mg/kg/h. This dosage has been shown to inhibit leukocyte activation and cytokine expression during ischemia and reperfusion and shock. In our experiment this dosage given intravenously had no significant improving effect on hemodynamics (except blood pressure) during endotoxemia, whereas intraintestinal protease inhibition with FOY7 significantly improved hemodynamics. Data are presented as mean ± SD. *Significantly different when compared with baseline value on the same group. ySignificantly different when compared with SHAM.
Fig 5. Representative histology sections of the rat small intestine at 5 hours after endotoxin administration. Groups are the same as in Fig 1. Endotoxemia-induced mucosal damage, loss of the brush border with leukocyte infiltration in control groups with and without intestinal lavage (CON, LAV). Intestinal protease inhibition reduced intestinal damage during endotoxemia (FOY).
splanchnic ischemia. Instead, IPI attenuated shockinduced hypotension by reducing the level of inflammatory mediators in the plasma.18,19 The pathophysiology of splanchnic ischemia, however, differs from endotoxin-induced shock. In septic shock, endotoxin can serve as a direct activating factor. As an activator for leukocytes or endothelial cells, endotoxin may cause lowered perfusion and ischemia. During splanchnic ischemia, the biochemical identity of the main trigger mechanism for leukocyte activation is still unknown. New activators may be formed, quite independent of endotoxin. Thus it is interesting to examine whether IPI may also improve endotoxin-induced shock parameters. In the current model of endotoxemia, IPI did not interfere with hemodynamic parameters during the first 20 to 60 minutes, indicating that at the beginning of endotoxemia, intestinal proteases are
not the main source for hemodynamic alterations. At a later time, however, IPI reduced endotoxininduced hypotension, tachycardia, and hyperventilation, and to microvascular and hemodynamic improvement by attenuation of a delayed inflammatory process after the initial endotoxin administration. Inflammation seems to be a major cause for shock and multiple organ failure during sepsis. Inflammation is accompanied by a powerful form of activation that includes leukocytes, endothelial cells, and possibly other cell types.34 Leukocyte activation leads to pseudopod formation and capillary plugging with leukocyte migration across the endothelial wall, local tissue inflammation, and cell death.11 Inflammation is also accompanied by a series of pathophysiologic processes, which may lead to cardiopulmonary dysfunction, including hypotension, tachycardia, and hyperventilation. The attenuation of inflammation with IPI may be one of the major reasons for the improvement of the microvascular perfusion. The biochemical structure of the inflammatory mediators produced in the intestinal wall is the subject of our current investigations. From pancreatic homogenates we have identified leukocyte-
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activating factors with hydrophilic and hydrophobic characteristics and a range of molecular weights, some of them less than 3kD.4,21 Proteases themselves may be less involved in activation of leukocytes,21 supporting the hypothesis that proteases have more of a role as producers of inflammatory mediators. That IPI inhibits the formation of inflammatory mediators supports the hypothesis that such mediators may be predominantly produced within the wall of the intestine by pancreatic digestive enzymes. IPI also reduced endotoxin-induced microvascular leukocyte and endothelial cell activation, parenchymal cell death, and the production of IL-6. Sepsis is accompanied by intestinal ischemia and mucosal cell injury.35-38 Elevation of the mucosal epithelial permeability permits entry of pancreatic enzymes into the interstitium of the intestinal wall. Entry of enzymes into the submucosal interstitium may lead to generation of inflammatory mediators by proteolytic cleavage within the intestinal wall. Our findings support this hypothesis, because IPI served to reduce the generation of activating factors in the small intestine as well as mucosal damage during endotoxemia. Additional aspects of this hypothesis remain to be examined. It may be argued that intestinal endotoxin or intestine-derived cytokines may be blocked by IPI. Lavage of the intestine was less effective in preventing the systemic inflammation than was lavage combined with protease inhibition. We investigated the relation between IPI, endotoxin activity, and tumor necrosis factor alpha (TNF-a) levels during intestinal ischemia-induced shock (unpublished results). IPI did not reduce intestinal endotoxin activity. The levels of TNF-a in intestinal homogenates during shock were identical in IPI and control animals. Endotoxin activity and TNF-a levels did not correlate with the amount of leukocyte activation either in the intestine or in the plasma. Taken together, this evidence suggests that intestinal endotoxin and intestine-derived TNF-a may be less involved in the mechanisms by which IPI attenuates shock. We hypothesize that intestinal absorption of FOY and inhibition of leukocyte activation and adhesion,39 as well as reduction of cytokine levels,40 may in part account for the improved hemodynamics and decreased microvascular inflammation in the current experiments. However, IV-administered FOY at a dose known to reduce leukocyte activation28 did not improve hemodynamics during sepsis in our model. Only endotoxin-induced hypotension was partially attenuated in this group,
possibly as a result of the inhibitory effect of FOY on the constitutive and inducible nitric oxide synthase.41 Our results do not clarify whether FOY leaking into the extraintestinal tissue may, in part, have accounted for the effects seen in the FOY group. Further studies are required with other protease inhibitors. Intestine-derived oxygen radicals may be influenced by IPI. Recent data demonstrated that IPI did not affect either xanthine dehydrogenase to xanthine oxidase conversion or xanthine oxidase activity in the ischemic intestine. The addition of allopurinol to the lavage fluid during IPI did not increase the beneficial effect of IPI even though allopurinol inhibited the xanthine oxidase activity in the ischemic intestine.19 Systemic leukocyte activation and shock may be related more closely to protease activity than to the action of oxygen free radicals derived from xanthine oxidase. In conclusion, we provide here the first evidence to suggest that, in addition to endotoxin, intestinal proteases may be involved in the generation of intestine-derived inflammatory mediators in the plasma. These mediators may sustain systemic cell activation and produce organ injury after an endotoxin challenge. Intraintestinal pancreatic protease blockade may have clinical utility in septic shock patients. The authors wish to thank Mr William H. Loomis, UCSD Department of Surgery, for technical help with the IL-6 measurements, and Dr Tony Hugli, La Jolla Institute for Molecular Medicine, for his thoughtful suggestions regarding this study. FOYÒ was a gift from Ono Pharmaceutical.
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