1Departments of General and 2Experimental Surgery, University of Heidelberg, 69120 Heidelberg, Germany; and 3Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114
Submitted 14 May 2004 ; accepted in final form 2 August 2004
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ABSTRACT |
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adhesion molecules; endothelial cells; proteases; systemic inflammatory response syndrome
ICAM-1 (CD54), a member of the immunoglobulin superfamily, is expressed constitutively at a low extent on the surface of endothelial cells. On cell activation, ICAM-1 is upregulated and participates in the firm adhesion of leukocytes to endothelium and in transendothelial cell migration (39). In acute pancreatitis, ICAM-1 is known to be upregulated in the pancreas and lungs, and both onset and extent of ICAM-1 expression correlate with the severity of organ injury (9, 26, 41). Furthermore, genetically induced ICAM-1 deficiency or administration of monoclonal antibodies against ICAM-1 has been proven to be effective in the prevention and treatment of these complications (9, 25, 41).
Although various mediators have been implicated in the upregulation of ICAM-1 in inflammatory disease (7, 17, 30), it is yet unknown which factors are involved in this process in acute pancreatitis. Transcriptional activation of the adhesion molecules ICAM-1 and VCAM-1 expression was found when endothelial cells were incubated with ascites collected from animals with necrotizing pancreatitis (27). Recently, our laboratory (12) has demonstrated that Mac-1 (CD11b/CD18), the ligand to ICAM-1, is upregulated on neutrophils incubated with trypsinated serum, giving evidence that the serine protease trypsin induces changes in serum, which cause leukocyte activation in acute pancreatitis, possibly by complement activation. In the present study, we provide direct evidence for the central role of trypsin in the initiation of leukocyte migration and organ injury. Trypsin induces changes in serum that result in ICAM-1 expression on endothelial cells in vitro, upregulates ICAM-1 expression in the pancreas and lungs in vivo, and increases ICAM-1-dependent interaction between leukocytes and the endothelial lining.
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MATERIALS AND METHODS |
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Human umbilical vein endothelial cell incubations.
Human umbilical vein endothelial cells (HUVECs) were plated at a concentration of 3 x 105 cells/well in 96-well, flat-bottom, microwell plates (Nunclon; Fisher Scientific, Pittsburgh, PA) and grown to confluence at 37°C in a humidified incubator with 5% CO2-95% air. For incubations, 20 µl (or 50 µl) of serum subjected to limited trypsin digestion (see Limited trypsin digestion of serum) were added to HUVECs. HUVECs incubated with TNF- (400 and 1,000 U/l; Sigma, St. Louis, MO) or serum alone served as controls. Incubation was allowed to proceed for 3, 6, 9, 12, and 24 h at 37°C. After incubation, the supernatant was withdrawn from endothelium layers and transferred to separate Nunclon microwell plates for measurement of soluble ICAM-1. Membrane-bound as well as soluble ICAM-1 were measured quantitatively by ELISA.
Cells and cell culture. HUVECs, EBM-2 endothelial growth cell medium, and EGM-2 supplement were purchased from Clonetics (San Diego, CA). HUVECs were grown on 100 x 20-mm tissue culture-treated dishes (Corning, Corning, NY) until 7090% confluence and then subcultured onto flat-bottom microwell plates. The plates were further incubated in the humidified incubator for 13 days to allow cell adherence and growth to desired density. Cells between passages 5 and 12 were used for all experiments.
Limited trypsin digestion of serum. Non-heat-inactivated bovine serum was subjected to limited trypsin digestion by incubation with bovine trypsin (final concentration 106104 M, n = 5 samples per group) and simultaneous addition of soybean trypsin inhibitor twice the concentration of trypsin (both from Sigma). Incubations were performed for 20 min at room temperature. Unlike incubation of serum with trypsin alone, limited trypsin digestion has previously been shown to effectively activate complement components in human serum and to induce neutrophil activation (33). Significant residual trypsin activity is not present in these serum preparations as measured spectrophotometrically using p-toluenesulfonyl-L-arginine methyl ester (Sigma, St. Louis, MO) as substrate (12). After completion of incubation, trypsinated serum was frozen and stored at 80°C until being used for HUVEC incubations.
Cellular ELISA for ICAM-1. The expression of ICAM-1 on HUVECs incubated with serum subjected to limited trypsin digestion was quantified by using a modified ELISA assay as described previously (24). Briefly, after completion of the incubation protocol and subtraction of the supernatant, cells were washed with PBS/Tween buffer (PBST), fixed with 1% paraformaldehyde (Sigma) for 30 min, and washed again with PBST. Nonfat dry milk (5%) was added to reduce nonspecific binding. Cells were incubated with 100 µl of monoclonal anti-human ICAM-1 (CD54; Pharmingen, San Diego, CA), diluted 1:1,000 in culture medium for 45 min, washed 5 times with PBST, and incubated in the dark with 100 µl of biotinylated anti-mouse secondary antibody (Vector Laboratories, Burlingame, CA) for 30 min. Subsequently, cells were washed as above and incubated in the dark with 100 µl streptavidin horseradish peroxidase (DAKO, Carpinteria, CA) for 30 min. After another washing with PBST, 100 µl of TMB peroxidase substrate (tetramethyl benzidine dihydrochloride K-Blued MAX; Neogen Lexington, KY) was added for 1530 min. The reaction was halted by the addition of 1 M sulfuric acid (50 µl/well). All incubations were carried out at room temperature. The expression of ICAM-1 was determined by the difference in absorbance at wavelength 450 vs. 630 nm measured by an automated microplate reader.
An ELISA-based cell count was performed to correct for possible differences in the amount of HUVECs in the wells after incubation with trypsinated serum or TNF-. After the cellular ELISA for ICAM-1, cells were washed in tap water. Crystal violet (0.08%, Sigma) was added for 5 min, followed by another thorough wash in tap water. Acetic acid (33%) was used to solubilize the nuclear stain. Optical density was read at 570 nm, representing the actual cell count per well.
Likewise, soluble ICAM-1 was determined in the supernatant of incubated HUVECs. After completion of incubations, the supernatant was transferred to separate 96-well Nunclon plates. Plates were kept overnight at 4°C to allow binding of protein to the wells. ELISA was then performed as described in Cellular ELISA for ICAM-1, starting with the addition of nonfat dry milk to reduce nonspecific antibody binding. Results of soluble ICAM-1 are expressed as the percentage of ICAM-1 in the supernatant at baseline.
In Vivo Experiments
The early effects of trypsin released into the systemic circulation in acute pancreatitis were imitated in the present study by intravenous infusion of trypsin into healthy rats. Previously, it has been shown by our laboratory (12, 15) that infusion of trypsin, as well as infusion of trypsinated serum into rats, results in pancreatic and pulmonary injury similar to that found in acute pancreatitis. Unlike in vitro incubations, which necessitate serum subjected to limited trypsin digestion (see Limited trypsin digestion of serum), the presence of serum containing plenty of antiproteases in the rat allows the use of trypsin infusion for in vivo experiments.
Experimental protocol. Bovine trypsin (Sigma) was infused intravenously into rats (n = 6) for 6 h at 60 nmol·kg1·h1. At this concentration, trypsin causes significant pancreatic and pulmonary injury, as assessed by the wet-to-dry ratio, myeloperoxidase activity, and histology (15). Infusions were divided into 3 aliquots/h, and each aliquot was freshly reconstituted in saline before injection to minimize autodigestion of trypsin. Saline was infused into control animals. At 6 h after the start of infusions, leukocyte-endothelium interaction and capillary blood flow were assessed in the head of the pancreas by intravital microscopy. In additional animals infused with trypsin, ICAM-1 expression in the pancreas and lungs was measured by Western blot analysis. Pancreatic and pulmonary leukocyte sequestration was assessed by activity of myeloperoxidase, an enzyme stored in the azurophilic granules of neutrophils. To evaluate whether changes in leukocyte-endothelial interaction induced by trypsin are related to ICAM-1 upregulation, one group of animals (n = 6) was pretreated with purified anti-rat ICAM-1 antibody (1.0 mg/kg; Pharmingen) before trypsin infusions were started.
Animals. Inbred male Wistar rats weighing 250300 g were used for experiments. Care was provided in accordance with the German law for care and use of laboratory animals. The study was approved by the Regierungspräsidium Karlsruhe, Germany, committee on animal care. Animals were fasted overnight before the experiments but allowed free access to water.
Anesthesia and catheter placement. Surgical anesthesia was induced by intraperitoneal injection of pentobarbital sodium (10 mg/kg, Nembutal; Sanofi-Ceva, Genova, Germany) and intramuscular injection of ketamine (40 mg/kg Ketanest 50; Parke-Davis, Berlin, Germany). Two polyethylene catheters (ID, 0.5 mm) were inserted into the left carotid artery and the right internal jugular vein, respectively, tunneled subcutaneously to the suprascapular area, and exited through a steel tether that allowed the animals free movement. The venous catheter was used for infusion regimens, and the arterial line was used for blood sampling and hemodynamic monitoring.
Intravital microscopy. Microcirculatory changes of the pancreas caused by trypsin infusion were assessed by intravital microscopy as described in detail elsewhere (16). Briefly, after a midline laparotomy was performed, the pancreas with the duodenal loop was gently exteriorized and placed in an immersion chamber containing Ringers lactate maintained at 37°C. The pancreatic microcirculation was then evaluated in epi-illumination using a fluorescence microscope (Fa. Leitz, Wetzlar, Germany). Therefore, animals were injected with FITC-labeled erythrocytes (0.5 ml/kg, hematocrit 50%) for capillary blood flow measurements and Rhodamin 6G (bolus of 0.1 ml) for in vivo staining of leukocytes and quantitation of leukocyte-endothelium interaction. Mean capillary red blood cell velocity was analyzed in four different regions of the pancreas in each rat. The mean red blood cell velocity in each area was calculated by averaging the velocity of erythrocytes in 15 ± 2 capillaries. According to their interaction with the endothelial lining, adherent and rolling leukocytes were assessed in postcapillary venules with a diameter of 2540 µm. Adherent leukocytes (stickers) were defined as cells that did not move or detach from the endothelium within the observation period of 30 s (10). Rolling leukocytes (rollers) were defined as those white cells moving at a velocity less than two-thirds of that of erythrocytes in the centerline of the venule (42). Both stickers and rollers were expressed as the number of cells per square millimeter of vessel surface, calculated from the diameter and length (100 µm) of the vessel segment studied. Off-line analysis of video recordings was performed in a blinded fashion using a computer-assisted microcirculation analysis system (Cap Image; Dr. H. Zeintl, Heidelberg, Germany).
Gel electrophoresis and Western blot analysis. Pancreas and lung tissue were homogenized in sodium phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide (Sigma) and 5% soybean trypsin inhibitor at a tissue-to-buffer ratio of 1:1.5. Protein concentration was determined by protein assay (Pierce, Perbio Science Deutschland, Bonn, Germany) and adjusted to a final protein concentration of 1.8 mg/ml. Electrophoresis was performed by SDS-PAGE. A total of 30 µl of tissue sample per well was loaded on the gel in nonreducing sample buffer. After electrophoresis, the proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad, München, Germany). The blotted membrane was blocked with nonfat dry milk (5%) in TBS/Tween-buffer (TBST) and agitated for 2 h with a mouse anti-rat ICAM-1 antibody (1:100 dilution in TBST; Serotec, Düsseldorf, Germany). After samples were washed with TBST, the secondary reagent, goat anti-mouse IgG linked to horseradish peroxidase (1:10,000 dilution, Linaris, Wertheim-Bettingen, Germany) was added. After repeated washing with TBST, the membrane was rinsed with water. The method of enhanced chemiluminescence (ECL Plus; Amersham Biosciences, Freiburg, Germany) was chosen for detection, according to the manufacturer's instruction. All incubations were carried out at room temperature.
Myeloperoxidase activity. Excised pancreatic and pulmonary tissues were rinsed with saline, blotted dry, snap frozen in liquid nitrogen, and stored at 80°C until thawing for determination of myeloperoxidase activity as previously described (15).
Statistical Analysis
Data are presented as means ± SE. One-way ANOVA was used for evaluation of differences between groups (when >2), followed by post hoc comparison of the means of the groups. Student's t-test was used for pairwise comparison of normal, distributed parameters. Values of P < 0.05 were considered significant.
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RESULTS |
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ICAM-1 expression on HUVECs. The expression of ICAM-1 on HUVECs incubated with trypsinated serum was significantly increased compared with controls incubated with untreated serum. Levels of ICAM-1 expression increased with higher concentrations of trypsin used for serum trypsination (Fig. 1). Highest levels of ICAM-1 expression were found at 9 h after the start of incubations. Larger amounts of trypsinated serum used for serum incubations (50 instead of 20 µl) did not further increase ICAM-1 expression. However, peak levels of ICAM-1 occurred earlier (data not shown).
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Similar to membrane-bound ICAM-1, significant amounts of soluble ICAM-1 were found in the supernatant of HUVECs stimulated with trypsinated serum. Again, levels of soluble ICAM-1 increased with higher amounts of trypsin used for serum trypsination (Fig. 2). When trypsin was used at a concentration of 104 M, levels of soluble ICAM-1 peaked at 9 h of HUVEC incubation but decreased significantly thereafter. HUVECs incubated with TNF- showed highest levels of soluble ICAM-1 at 12 h. Unlike incubations with trypsinated serum, soluble ICAM-1 levels remained elevated for the entire observation period of 24 h (Fig. 2).
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In confirmation of our in vitro findings, Western blot analysis demonstrated higher levels of ICAM-1 expression in the lungs of animals injected with trypsin compared with control animals injected with saline only (Fig. 3). ICAM-1 expression was more pronounced in the lungs than in the pancreas. Likewise, animals showed significant organ injury with significant leukocyte sequestration in the pancreas and lungs as determined by myeloperoxidase activity (pancreas: 13.3 ± 1.9 vs. 6.1 ± 0.9 U/mg; lungs: 112.2 ± 9.7 vs. 47.8 ± 7.2 U/mg; both trypsin vs. control). This is in agreement with previous studies (15).
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DISCUSSION |
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In agreement with these in vitro findings, we demonstrated that ICAM-1 expression is also increased by trypsin in vivo. This increase of ICAM-1 expression was more pronounced in the lungs compared with the pancreas, possibly because of the extended pulmonary microvasculature. Associated with the increased ICAM-1 expression in trypsin-infused animals was the development of organ injury, markedly of the lungs. Similar to pancreatitis-associated lung injury, this injury is of a neutrophil-dependent character (15). Applying intravital microscopy, a method that allows the investigation of leukocyte-endothelium interaction and microvascular perfusion of organs in real-time conditions in vivo, we have found convincing evidence that the observed trypsin-induced changes in adhesion molecule expression are relevant for the process of leukocyte migration. In the pancreas of animals infused with trypsin, the amounts of rollers and stickers were markedly increased compared with controls. Infusion of an antibody against ICAM-1 resulted in the amelioration of these changes. These findings support the hypothesis that the upregulation of ICAM-1 by proteases released from the pancreas may also be relevant in the pathogenesis of pancreatitis-associated organ injury.
In acute pancreatitis, ICAM-1 is upregulated in both the pancreas and the lungs, and the extent of ICAM-1 expression correlates with the severity of organ injury (26, 41). In previous studies (11, 15, 40), we have shown that the occurrence and severity of pancreatitis-associated lung injury corresponds to the levels of circulating trypsinogen and its activation to trypsin and that neutrophils are involved in the development of this injury. Furthermore, it has been demonstrated that the expression of Mac-1 (CD11b/CD18), the ligand to endothelial ICAM-1, is increased on the surface of neutrophils incubated with trypsinated serum (12). Mac-1, a membrane-bound adhesion molecule of the integrin group, is primarily expressed on the surface of neutrophils on stimulation. There is general agreement that the firm adhesion of neutrophils to the endothelial lining is, at least in part, mediated by the interaction of ICAM-1 and Mac-1 (4). With the indirect trypsin-induced upregulation of both Mac-1 and ICAM-1, we have identified trypsin as a mediator capable of inducing neutrophil and endothelium activation, followed by extravasation of cells and manifestation of organ injury in acute pancreatitis.
Serine protease trypsin has been assumed for a long time to cause local and distant organ injury in acute pancreatitis. Whereas intra-acinar and interstitial trypsinogen activation are widely accepted to be central events in the early pathogenesis of acute pancreatitis (8, 13, 37), the role of trypsin in the pathogenesis of distant organ injury is controversial. Although selective trypsin infusion caused lung injury in several animal models (5, 15, 38), systemic trypsin has been questioned as playing a significant role in acute pancreatitis due to its quick binding to protease inhibitors. In the present study, we demonstrate that trypsin-induced changes in serum can induce adhesion molecule expression and thereby initiate leukocyte migration into tissue. The limited trypsin digestion of serum, as applied in our study, included the simultaneous addition of soybean trypsin inhibitor twice the concentration of trypsin. Unlike trypsin alone, using this procedure has also been demonstrated as inducing neutrophil activation with subsequent induction of the respiratory burst (33). It may closely reflect in vivo conditions in which protease inhibitors are present in excess.
Recently, several studies (1, 27) have focused on the regulation of adhesion molecules in acute pancreatitis. Confirming our results, increased expression of Mac-1 or ICAM-1 was found on neutrophils and endothelial cells incubated with serum or ascites from animals with acute pancreatitis. The proinflammatory substances that are possibly inducing this cell activation include proteases other than trypsin, cytokines, or complement proteins. Interestingly, complement activation has been demonstrated by trypsin (2, 28, 33) and also in acute pancreatitis (18, 34). Therefore, complement proteins may mediate the trypsin-induced leukocyte activation and thereby contribute to the development of systemic organ injury in acute pancreatitis (12).
Most of the membrane-bound adhesion molecules are also known to exist in a soluble form. Soluble ICAM-1 was first described in 1991 (32, 35) in the blood of healthy human subjects. Subsequently, a number of studies (6, 20, 29) reported increased levels of soluble ICAM-1 in inflammatory disease, but little is known about soluble ICAM-1 in acute pancreatitis. Our in vitro experiments showed elevated levels of soluble ICAM-1 in the supernatant of HUVECs activated by trypsinated serum, and soluble ICAM-1 increased with higher amounts of trypsin used for serum trypsination. This finding is in agreement with the proposal that soluble adhesion molecules may reflect endothelial cell activation and the magnitude of expression of their membrane-bound counterparts. Whether the circulating levels of soluble ICAM-1 are useful markers or predictors for the severity of acute pancreatitis, as suggested by recent studies (23, 31), remains to be investigated.
In summary, we have demonstrated that trypsin induces changes in serum that result in upregulation of ICAM-1 and in leukocyte migration. Inhibition of ICAM-1 expression may be a potential therapy of systemic complications in acute pancreatitis. A diagnostic tool may be available by measurement of soluble ICAM-1 in serum of patients.
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GRANTS |
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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