Over the last several years it has become apparent that endothelial cells are active participants in the regulation of organ inflammation as well as blood flow and coagulation (1,2). As regulators of coagulation and inflammation, endothelial cells in conjunction with circulating neutrophils, are able to promote tissue ischemia and injury. In fact, endothelial-leukocyte interactions resulting in tissue injury appear to be a common pathway by which a diverse number of initiating factors leads to multiple organ failure (3). Adhesion molecules play a pivotal role in endothelial cell-leukocyte interactions resulting in leukocyte migration from the intravascular space into the interstitial space. These adhesion molecules include the selectins, integrins, and intracellular adhesion molecules (ICAM). E-Selectin and P-selectin are members of the selectin family of adhesion molecules. These selectins function in the initial step of recruitment of leukocytes to the site of an inflammatory reaction. E-selectin is an inducible endothelial cell-specific surface glycoprotein, which mediates the binding of neutrophils and other inflammatory cells to the endothelium. The expression of E-selectin on the surface of endothelial cells can be induced by treatment with tumor necrosis factor-α (TNF-α) and bacterial lipopolysaccharide (4). P-selectin is expressed on the surface of activated endothelial cells and platelets. The preformed form of P-selectin is stored in the Weibel-Palade bodies of endothelial cells and in the alpha-granules of platelets and can be mobilized to the surface of activated endothelial cells within minutes. This rapid transport is thought to contribute to the early stage of rolling during leukocyte migration. The transcription-dependent form of P-selectin has been shown to be synthesized in response to endotoxin (5) or TNF-α (6). In contrast to the selectins, the ICAM-1 molecule is a member of the immunoglobulin supergene family and is present at low levels on unstimulated endothelial cells. ICAM-1 interacts with integrins located on the leukocyte cell surface mediating the firm adhesion of neutrophils to the endothelial cell. The expression of ICAM-1 is upregulated in response to TNF-α, interleukin (IL)-1, interferon-gamma, or LPS (7).
Endothelial cells also produce a plethora of cytokines that are involved in the fine-tuning of the endothelial response to tissue injury. IL-6 is a cytokine produced by a wide variety of cell types that can increase the permeability of endothelial cells (8). Recently, it has been demonstrated that IL-6 is an important autocrine factor produced by endothelial cells that contributes to the increase in endothelial permeability during hypoxia (9).
Recent studies have demonstrated that gut-derived inflammatory factors play an important role in causing multiple organ dysfunction during trauma/hemorrhagic shock (T/HS) and sepsis (10). Recently, we have documented that that the lymphatic route is the primary route of egress for gut-derived toxic factors produced by the injured or ischemic gut. This became evident by demonstrating that ligation of the mesenteric lymph duct prior to T/HS protects against shock-induced lung injury in the rat (11,12). Furthermore, these studies have also demonstrated that lymph duct ligation prevents shock-induced P-selectin upregulation in the lung (13). This observation suggests that inflammatory factors carried in the lymph are contributing to the lung injury by a mechanism involving the upregulation of adhesion molecules. To confirm this hypothesis, in the present study we investigated the effect of T/HS mesenteric lymph from rats on the cell-surface expression of E-selectin, P-selectin, and intracellular adhesion molecule-1 (ICAM-1) in cultured human umbilical vein endothelial cells (HUVECs). Furthermore because, IL-6 is an important contributor to the hypoxia-induced derangement of endothelial barrier function (9), we hypothesized that T/HS lymph may induce an increase in endothelial permeability by triggering the release of IL-6.
MATERIALS AND METHODS
HUVECs were obtained from BioWhittaker and cultured in endothelial growth medium (BioWhittaker, MD, USA). The HUVECs used for all experiments were from a Caucasian female umbilical cord. The cells were grown in a humidified atmosphere of 95% air and 5% CO2, at 37°C. Second to fourth passage HUVECs were used in all experiments.
Adult male specific pathogen-free Sprague-Dawley rats (Charles River Laboratories, Portage, MI) weighing 350–450g were used after a minimum seven-day acclimation period. The animals were housed under barrier conditions and kept at 25°C with a 12-h light/dark cycle. Rats were allowed free access to water and chow (Teklad 22/5 Rodent Diet W-8640, Harlan Teklad, Madison, WI). All animals were maintained in accordance with the recommendations of the “Guide for the Care and Use of Laboratory Animals”, and all experiments were approved by the New Jersey Medical School Animal Care and Use Committee.
Experimental protocol for T/HS and trauma/sham shock (TSS)
The rats were anesthetized with sodium pentobarbital (50 mg/kg) injected intraperitoneally (i.p., Veterinary Laboratories, Inc. Lenexa, Ka) and the right femoral artery was isolated by minimal dissection and aseptically cannulated with polyethylene (PE-50) tubing containing 0.1 mL of heparinized saline. The catheter was connected in-line to a blood pressure recorder and polygraph (Grass Model 79E Data Recorder, Quincy, MA), to allow continuous blood pressure monitoring. Using aseptic technique, the right external jugular vein was cannulated with a 50-gauge silicone catheter containing 0.1 mL of heparinized saline. A laparotomy was performed and the mesenteric lymphatic duct was aseptically cannulated with silicone tubing that exited the right flank. An additional polyethylene (PE-90) catheter placed through a left upper quadrant stab wound was passed through the stomach and into the duodenum. This gastrostomy catheter was secured in place with cyanoacrylate glue (Loctite Corp. Hartford, CT) and used to deliver a constant infusion of normal saline at a rate of 3.9 mL/hr using a syringe pump (Model 341B, Orion Research Inc, Boston, MA). The abdomen was subsequently closed in two layers using a running 4.0 silk suture. To induce shock, blood was withdrawn from the jugular vein into a syringe containing 10 units of heparin suspended in 0.3 mL of 0.9% normal saline to prevent clotting. The blood pressure was reduced to 30mmHg and maintained at this level for 90 min by the careful withdrawal or re-infusion of shed blood (kept at 37°C) as needed. Mesenteric lymph samples were collected in sterile, heparin-wetted, 1mL tuberculin syringes on ice. After resuscitation, the animals were placed into Bollman-type restraining cages (Stoelting Co., Wood Dale, IL) to avoid the use of additional anesthesia and lymph collection continued in hourly increments. Thus, mesenteric lymph samples were collected for 1 h prior to shock, during the 90 min shock period and hourly for 6 additional hours post resuscitation. At the end of the sixth hour after resuscitation, the animals were anesthetized with an additional i.p. dose of pentobarbital and sacrificed. Portal blood was collected in syringes containing 10 units of heparin and centrifuged (1000 g for 20 min at 4°C) to obtain plasma, while lymph was centrifuged at 500 g for 15 min at 4°C to separate the humoral from the cellular components and aliquots were immediately frozen and stored at −80°C. T/SS rats were anesthethized and their mesenteric lymph duct was cannulated. However, no blood or fluid was withdrawn or infused.
Cell surface enzyme-linked immunoassay (ELISA) for the determination of adhesion molecule expression (14) and cell viability assay
HUVECs were seeded at 20,000 cells per well onto biocoat matrigel 96 well plates (Becton Dickinson Bedford, MA) and used for the enzyme immunoassay protocols. To test the effect of T/HS lymph on HUVEC adhesion molecule expression, cells were treated with 3% pre-T/HS lymph, or T/HS lymph collected at 2 or 3, or 6 h after T/HS. The following controls were used: 3% portal vein plasma from T/HS rats, and 3% pre- and post-T/SS lymph. The HUVECs were incubated with the lymph or plasma samples for 4 h at 37°C. The 4-h time point was chosen because previous studies have shown that various proinflammatory stimuli such as TNF-α and LPS induce both E- and P-selectin, as well as ICAM-1 expression in HUVECs 4 h after stimulation (15,16). At the end of the incubation period, the supernatant was removed and the cells were washed twice with phosphate buffered saline (PBS) and fixed with 1.5% paraformaldehyde for 30 min. Subsequently, the cells were blocked with 2% bovine serum albumin, and then incubated for 1 h at 37°C with a monoclonal antibody to E-selectin, P-selectin, or ICAM-1 (BD PharMingen, San Diego, CA). Then, the cells were incubated with an alkaline phosphatase conjugated secondary antibody for 1 h. The cells were again washed three times following which p-nitrophenyl disodium phosphate (1mg/mL, Sigma, St. Louis, MO) was added and incubated at room temperature for 30 min. The colorimetric reaction was then read in a plate reader at 410 nm.
Because post-shock mesenteric lymph collected at 2 or 3 h after shock has significant endothelial cell toxicity, endothelial cell viability studies were performed using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,-diphenyl tetrazolium bromide)-based cell cytotoxicity kit obtained from Sigma (TOX 1; Sigma, St. Louis, MO). Since the number of viable HUVECs was different between the groups, cell viability was factored into the calculation of the adhesion molecule expressed. Adhesion molecule expression was calculated using the following equation:EQUATION
Measurement of soluble E selectin
For the detection of soluble E selectin, HUVECs were incubated for 24 h with 5% pre- and post-T/HS or T/SS lymph, medium, IL-1β, or TNF-α. Soluble E selectin was assayed in the supernatants collected at the end of the incubation period. Soluble E selectin concentrations were determined using an ELISA kit from R&D systems (Minneapolis, MN). Optical density was read on a microplate reader set at 450 nm.
HUVECs in 96-well plates were treated with 5% pre or post-shock T/HS lymph for 24 h. Human IL-6 levels were determined from the cell supernatants using commercially available ELISA kits (R&D, Minneapolis, MN) as previously described (17) and according to the manufacturer's instructions.
The Tukey-Kramer multiple comparison ANOVA test was used. The results are expressed as mean ± standard deviation or standard error of means of n = 6 wells. P values less than 0.05 are considered significant.
T/HS lymph decreases HUVEC viability
First we confirmed our previous observation (11,12,18) that post-T/HS lymph causes endothelial cell death. After a 4-h incubation of HUVECs with T/HS lymph collected 2 or 3 h after shock, cell viability decreased by 25% as compared to pre-T/HS lymph, 6-h post-T/HS lymph, portal vein plasma, T/SS lymph, or medium (Fig. 1).
P selectin expression is increased in response to post-T/HS lymph
Because mesenteric lymph duct ligation prior to T/HS prevents the shock-induced upregulation of P-selectin in the rat lung (13), first we examined whether T/HS lymph is able to upregulate the expression of P-selectin in HUVECs. As shown in Figure 2, pre-T/HS lymph, 6-h post-T/HS lymph, post-T/HS portal vein plasma, or T/SS lymph did not upregulate P selectin expression as compared to medium. On the other hand, incubation of HUVECs with 2 or 3 h post-T/HS lymph resulted in a >3 fold increase in P-selectin expression (Fig. 2).
Post-T/HS lymph induces the upregulation of cell surface E selectin expression in HUVECs
Next, we studied the effect of T/HS lymph on the expression of cell surface E-selectin in HUVECs. As observed for P-selectin, pre-T/HS lymph, 6-h post-T/HS lymph, post-T/HS portal vein plasma, or T/SS lymph did not upregulate E-selectin expression as compared to medium. However, incubation of HUVECs with 2 or 3 h post-T/HS lymph resulted in a >2.2 fold increase in E-selectin expression (Fig. 3).
Post-T/HS lymph does not induce the shedding of E selectin in HUVECs
Similar to previous studies (14), treatment of HUVECS with either TNF-α or IL-1β for 24 h induced the appearance of E selectin in the cell supernatants. However, neither the treatment of HUVECs with pre- or post-T/HS lymph, as well as T/SS lymph triggered the release of E selectin (Fig. 4).
Post-T/HS lymph induces the upregulation of cell surface ICAM-1 expression in HUVECs
Pre-T/HS lymph, 6-h post-T/HS lymph, post-T/HS portal vein plasma, or T/SS lymph did not induce the upregulation of ICAM-1 expression on HUVECs as compared to medium. On the other hand, incubation of HUVECs with 2 or 3 h post-T/HS lymph resulted in a >2.4 fold increase in ICAM-1 expression (Fig. 5).
Post-T/HS lymph augments IL-6 production by HUVECs
IL-6 levels in pre-T/HS-treated HUVECs were low and comparable to those in the medium controls (Fig. 6). However, post-T/HS lymph caused a significant increase in IL-6 production (Fig. 6).
Previous work in our laboratory has shown that after T/HS, gut-derived inflammatory factors play an important role in causing secondary lung injury characterized by an increase in lung permeability and neutrophil infiltration (11,12,18). A further important finding of these studies is that these detrimental gut-derived factors reach the lung via the mesenteric lymphatic system and not the portal circulation. Several lines of evidence are available to support this notion. First, ligation of the mesenteric lymph duct prevents lung injury in rats subjected to T/HS, as assessed by total protein content in bronchoalveolar lavage fluid, lung permeability to Evans blue dye, and alveolar apotosis (12). Second, mesenteric lymph obtained from rats subjected to T/HS but not T/SS increases endothelial cell monolayer permeability and causes endothelial cell death in vitro. Although the mechanism by which T/HS lymph injures the lung is not known, results from a previous study by our group suggested that increases in the expression of adhesion molecules may play a role in the harmful effects of T/HS lymph (13). In that study, we observed that mesenteric lymph duct ligation prevented the T/HS-induced upregulation of P-selectin in the lung. Because the upregulation of P-selectin and other adhesion molecules on the endothelial cell surface is an important process that contributed to neutrophil-mediated endothelial injury during ischemia/reperfusion (7), we hypothesized that the mesenteric lymph–induced lung injury after T/HS may involve such a mechanism. The results of the current study demonstrate that the expression of both P and E selectin, as well as ICAM-1 is augmented by T/HS lymph in endothelial cells in vitro. This observation suggests that T/HS lymph may, in fact, exert at least part of its deleterious effects on T/HS-induced lung injury by increasing the expression of adhesion molecules. The effect of T/HS lymph on adhesion molecules has a time course resembling that of the toxic effect of T/HS lymph on endothelial cells. That is, while the T/HS lymph collected 2–3 h after the induction of shock is capable of upregulating adhesion molecules and at the same time triggering cell death, the T/HS lymph obtained 6 h after shock loses its ability both to induce adhesion molecule expression and cell death (19). Interestingly, the time course of the cytotoxic and the adhesion molecule activating effects of T/HS lymph on HUVECs differs from its effect on HUVEC monolayer permeability, as T/HS lymph increases HUVEC permeability even when collected 6 h after shock (19). Thus, because of the difference in the time course of the different effects of T/HS lymph, it is likely that T/HS lymph contains several biologically active factors that exert their affects utilizing different mechanisms. However, it is also possible that there is only one such factor, whose concentration may decline with time. In such a scenario, the levels of this factor early after shock would be high enough to cause endothelial cell death and adhesion molecule upregulation, but at later time points, lower levels of this factor could only induce an increase in permeability. The fact that only the 2–3 h but not the 6 h post-T/HS lymph was able to upregulate the expression of adhesion molecules under the in vitro conditions could suggest that other factors than those originating from the lymph may contribute to the continuous presence of neutrophils in the lung observed after T/HS. However, it is also possible that the upregulation of adhesion molecules caused by the early, 2–3 h post-T/HS lymph is long lasting. That is, once they are induced they remain expressed for a longer period. This idea is supported by the observation that the increase in adhesion molecule expression on endothelial cells induced by TNF-α can be detected even 24 h after the stimulus (15,16).
At this point, it would be premature to speculate on what the different factors that cause endothelial cell damage and activation are. We have recently obtained evidence that the putative factor causing cell death is larger than 100,000 d, suggesting that cytokines, which are smaller than 100,000 d are not responsible for the toxic effect of T/HS lymph (20). However, since the toxic and activating factors may be different, the possibility that cytokines may account for the activating effects of T/HS lymph can not be ruled out. On the other hand, it is worth noting that in contrast to the effect of TNF-α or IL-1β, T/HS lymph does not induce the shedding of E selectin from endothelial cells. This observation suggests that cytokines and T/HS lymph activate HUVECs by different mechanisms. Finally, it is noteworthy that because bacterial endotoxin is a well-known inducer of adhesion molecule expression in HUVECs, it could be an ideal candidate for being the factor causing the activating effects of lymph. However, we have previously shown that T/HS lymph does not contain significant amounts of endotoxin (20). Therefore it is unlikely that endotoxin is involved in the stimulatory effects of T/HS lymph on endothelial adhesion molecule expression.
While the upregulation of adhesion molecules caused injury by an indirect mechanism involving neutrophils, soluble factors produced by endothelial cells, such as IL-6 can directly increase endothelial permeability in vitro (8). Furthermore, an in vivo study using IL-6 knockout animals demonstrated that IL-6 plays an essential role in lung PMN infiltration following HS. Our data showing that T/HS lymph augments the production of IL-6 by endothelial cells suggests that this cytokine may be an autocrine factor that could account for some of the harmful effects of T/HS lymph. It is important to point out at this point that the increase in IL-6 levels in the T/HS lymph-treated HUVEC supernatants does not originate from the T/HS lymph itself, because the IL-6 assay used in our study is specific for human IL-6. Furthermore, previously we demonstrated that T/HS lymph IL-6 levels are comparable to those found in T/SS lymph (21). Finally, although we did not measure other cytokines in the T/HS lymph-treated HUVECs, it is possible that IL-1β and/or TNF-α, both of which can increase HUVEC permeability, is induced by T/HS lymph.
The findings of the present in vitro study confirm our in vivo observation that gut-derived factors contained in the mesenteric lymph contribute to the upregulation of pulmonary endothelial cell adhesion molecules after T/HS (13). In summary, the upregulation of both E- and P-selectin expression, as well as ICAM-1 expression and IL-6 production in response to T/HS lymph in endothelial cells demonstrates that T/HS lymph contains factors that induce a change in the endothelium phenotype from a noninflammatory to a proinflammatory procoagulant one.
This study was supported by NIH Grant GM59841 (Dr. Deitch)
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