Hydroxyethyl starch (HES) solutions are modified natural polymers of amylopectin. They are often used as plasma expanders, in part because of their therapeutic safety, stable effects on plasma volume, and few associated incidences of anaphylactic reactions. Some animal studies imply that fractionated HES solutions with a molecular range of 100–1000 kDa may be capable of plugging leaky capillaries in inflammatory states (1,2). This potential effect of HES has recently been demonstrated in trauma patients (3). However, there are also studies with contradictory results (4). Therefore, additional studies need to be performed to clarify this protective effect of HES, and, if possible, explore the potential mechanisms that have not yet been interpreted.
Endotoxemia is always accompanied with systemic inflammatory response syndrome, which induces multiple organ failure, including the lung, liver, and kidney. Microvascular injury and increased vascular endothelial permeability are its characteristic features. One crucial event that leads to this injury is neutrophil activation followed by neutrophil infiltration and accumulation in various organs. Bacterial endotoxin (lipopolysaccharide; LPS) can induce the expression of adhesion molecules (E selectin, P selectin, and intercellular adhesion molecule-1 [ICAM-1]) on endothelial cells; activate and upregulate integrins (CD11b/CD18) on the neutrophil cell surface (5); cause the expression of chemokines, including interleukin (IL)-8 and cytokine-induced neutrophil chemoattractant (CINC) in several constitutive cells and resident macrophages (6,7); and release other proinflammatory cytokines (tumor necrosis factor (TNF)-α and IL-1) (8). These mediators then act in concert to promote neutrophil activation and migration into the interstitium, where neutrophils cause endothelial cell and tissue damage by releasing elastase, cathepsin G, and oxygen free radicals, leading to capillary leak and tissue edema (9).
Nuclear factor-κB (NF-κB) is an important regulation factor that plays an essential role in the transcriptional induction of cell adhesion molecules, chemokines, proinflammatory cytokines, and enzymes that contribute to endothelial damage and development of multiorgan injury (10). NF-κB can be induced by LPS in many organs and is increased in neutrophils in endotoxemia (11).
No studies have investigated the effects of HES on capillary permeability in acute endotoxemia. In this study, we used a third-generation HES with a medium molecular weight and a low degree of substitution (HES 200/0.5) to determine its effect on lung capillary permeability in endotoxic rats, the effects on lung neutrophil accumulation, expression of CD11b on the blood neutrophil cell surface, lung CINC protein level, and NF-κB activation in blood neutrophils and lungs, exploring the possible mechanisms underlying its reduction in capillary leak.
Male Wistar rats weighing 250–300 g were purchased from the Animal Center of the Chinese Academy of Science, Shanghai, China. The rats were fed rat chow with free access to tap water and housed in temperature- and humidity-controlled animal quarters with a 12-h light/dark cycle. All procedures were approved by the Institutional Animal Care Committee.
Animals were anesthetized with urethane (1250 mg/kg intraperitoneally [IP]). A polyethylene catheter was implanted in the right external jugular vein for the continuous infusion of solutions by using a Razel Model WZ-50C syringe pump. The rats were randomly divided into seven groups (six rats per group): 1) controls, 2) LPS; 3–6) LPS plus HES 3.75, 7.5, 15, or 30 mL/kg; and 6) HES alone (30 mL/kg). Immediately after the time reading, LPS (6 mg/kg IP; Escherichia coli O55:B5, Sigma Chemical Co., St. Louis, MO) was given over 20 s. HES (HAES-Steril 200/0.5, 6%; Fresenius Kabi) was infused beginning at 1 min at 0.2 mL/min. In the control and HES-alone groups, 0.9% saline vehicle (3 mL/kg IP) was given instead of LPS at Time 0. In the control and LPS groups, saline 30 mL/kg was infused instead of HES. In a pilot study, the blood pressure of rats was measured with a microtip manometer (Millar, Houston, TX) inserted into the femoral artery; no significant hemodynamic instability was encountered during the procedure.
The rats were killed by exsanguination 4 h after the LPS challenge, and lung microvascular permeability was assessed by quantitating the extravasation of Evans blue dye into lung parenchyma. In another set of experiments, animals underwent the same treatments. Heparinized blood samples were obtained by a cardiac puncture with a heparin-coated 18-gauge needle either at 2 h (for neutrophil isolation and electrophoretic mobility shift assay [EMSA]) or 4 h (for flow cytometry analysis) after LPS challenge. The lung tissue was also collected either at 2 h (for EMSA) or 4 h (for determination of lung wet/dry weight ratio, myeloperoxidase [MPO] analysis, and enzyme-linked immunosorbent assay [ELISA]) after LPS challenge, frozen in liquid nitrogen, and stored at −80°C.
Lung capillary permeability was assessed with the Evans blue dye extravasation method (12). Briefly, animals were injected with 2% Evans blue (20 mg/kg; Sigma Chemical Co.) via the jugular vein 15 min before killing. After the rats were killed, the lungs were removed, and the wet weight was determined. The dye was then extracted from the tissue by incubation with 4 mL of formamide for 24 h at 37°C. The quantity of dye extracted was determined spectrophotometrically at 620 nm and calculated from a standard curve established with known amounts of Evans blue dye. Results are expressed as milligrams of dye per gram of wet tissue.
To determine the lung wet/dry weight ratio, lung tissue samples were taken after the rats were killed. Excess fluid was blotted from specimens, and wet weights were measured immediately. Dry weights were measured after drying specimens at 80°C for 72 h to constant weight. The lung wet/dry weight ratio was then calculated.
Lung MPO activity was determined as an index of tissue neutrophil accumulation. To measure tissue MPO activity, frozen lungs were thawed, and MPO was extracted by homogenization and sonication, as described previously (13). MPO activity in the supernatant was measured and calculated from the absorbance (at 460 nm) changes resulting from decomposition of H2O2 in the presence of o-dianisidine. The lung tissue CINC content was measured by an ELISA with a Rat GRO/CINC-1 immunoassay kit (Amersham, UK) according to the manufacturer’s instructions.
Blood samples from each animal collected at 4 h after the LPS challenge were prepared for cytometric analysis. After the lysing procedure, the number of leukocytes per sample was counted. Then 5 × 106 cells were incubated with a phycoerythrin-labeled mouse anti-rat CD11b antibody (Serotec, Oxford, UK) on ice for 30 min. After samples were washed, they were cold-centrifuged, and the cell pellet was resuspended in 500 μL of phosphate-buffered saline. The cells were then read on a Becton-Dickinson FACSCalibur. Negative controls were incubated with phycoerythrin-labeled mouse immunoglobulin G2a (Serotec).
Neutrophils were isolated by using Ficoll-Paque gradient centrifugation and dextran sedimentation. After hypotonic lysis of residual red blood cells, neutrophils were collected. Viability, as determined by trypan blue exclusion, was consistently >95%. Neutrophil purity, as determined by Wright’s staining cytospin preparations, was >98%. Neutrophils were then stored at −80°C.
Nuclear extracts of the lung tissues and neutrophils were prepared by hypotonic lysis followed by high salt extraction. Briefly, ∼0.1 g of frozen lungs or separated neutrophils was homogenized in 0.5 mL of ice-cold Buffer A, composed of 10 mM HEPES pH 7.9, 10 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonyl fluoride (all from Sigma Chemical Co.). The homogenates were centrifuged for 30 s at 500 g at 4°C to eliminate any unbroken tissue. The supernatants were incubated on ice for 20 min, vortexed for 30 s after the addition of 50 μL of 10% Nonidet P-40 (Sigma Chemical Co.), and then centrifuged for 1 min at 5000 g at 4°C. The crude nuclear pellet was suspended in 200 μL of ice-cold Buffer B (20 mM HEPES pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, and 25% vol/vol glycerol) and incubated on ice for 30 min, mixed frequently, and centrifuged at 12,000 g at 4°C for 15 min. The supernatants were collected as nuclear extracts and stored at −80°C for use. Protein concentration was determined by using a bicinchoninic acid assay kit with bovine serum albumin as the standard (Pierce Biochemicals, Rockford, IL).
EMSA was performed by using a commercial kit (Gel Shift Assay System; Promega, Madison, WI). The NF-κB consensus oligonucleotide probe (5′-AGTTGAG GGGACTTTCC CAGGC-3′) was end-labeled with [γ-32P]adenosine triphosphate (Free Biotech, Beijing, China) with T4-polynucleotide kinase. Nuclear protein (30 μg) was preincubated in a total volume of 9 μL in a binding buffer, which consisted of 10 mM Tris-HCl pH 7.5, 4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, and 0.05 mg/mL poly(di-dc) · poly(di-dc) for 10 min at room temperature. After the addition of the 32P-labeled oligonucleotide probe, the incubation was continued for 20 min at room temperature. The reaction was stopped by adding 1 μL of gel loading buffer, and the mixture was subjected to nondenaturing 4% polyacrylamide gel electrophoresis in 0.5× Tris-borate-EDTA buffer. The gel was vacuum-dried and exposed to radiograph film (Fuji Hyperfilm) at −70°C with an intensifying screen.
All data were expressed as mean ± se. Statistical significance was determined by one-way analysis of variance, followed by Tukey’s test; P < 0.05 was considered significant.
We used the Evans blue dye extravasation method to study the lung capillary permeability. Challenge with LPS caused a 2-fold increase in the microvascular permeability index in the lung (Fig. 1). Treatment of the LPS-challenged animals with HES 3.75 and 7.5 mL/kg, but not 15 and 30 mL/kg, significantly reduced (P < 0.05) the LPS-induced increase in permeability. HES alone had no effect on permeability index.
The wet/dry weight ratio, which represents the percentage of tissue water, is another index of tissue microvascular permeability. The results of this study were in parallel with those of the Evans blue dye study (Fig. 2). LPS significantly increased the lung wet/dry weight ratio. Rats co-treated with HES 3.75 and 7.5 mL/kg, but not 15 and 30 mL/kg, had decreased ratios compared with rats treated only with LPS. HES alone had no significant effect.
We studied the functional consequence of HES on neutrophil influx into the lungs by using MPO activity as an index of tissue neutrophil accumulation. As shown in Figure 3, MPO levels were significantly increased from 0.38 ± 0.013 U/g in control rats to 0.67 ± 0.029 U/g in the LPS group of rats and were reduced to 0.51 ± 0.015 U/g, 0.48 ± 0.026 U/g, 0.56 ± 0.040 U/g, and 0.60 ± 0.033 U/g in the rats treated with LPS plus HES 3.75, 7.5, 15, or 30 mL/kg. Control rats and rats treated with HES alone had similar lung MPO activities.
Increased surface expression of β2 adhesion molecules is important for neutrophil adherence to endothelial cells. As shown in Figure 4, LPS caused a more than twofold increase in surface expression of CD11b. Rats given increasing doses of HES progressively decreased the expression. Treatment of the LPS-challenged animals with HES 3.75, 7.5, 15, and 30 mL/kg reduced the LPS-induced increase of CD11b expression by 28%, 33%, 47%, and 50%, respectively. Treatment of control animals with HES did not affect the CD11b level on neutrophils.
CINC plays a pivotal role in neutrophil recruitment and acute lung injury. To address whether HES affects lung CINC expression, we compared CINC protein levels in different groups. ELISA results showed low CINC protein levels in control lungs (Fig. 5); these levels increased by 2.5-fold in lungs of LPS-challenged animals. Treatment with HES 3.75, 7.5, and 15 mL/kg significantly reduced (P < 0.05) the LPS-induced increase in CINC levels. HES alone had no effect on CINC expression.
To investigate the possible mechanism of HES action, we performed the EMSA experiment to examine the effect of HES on the activation of NF-κB induced by LPS. We quantitated these NF-κB bands by using densitometry. As shown in Figure 6, the NF-κB activity was low in nuclear protein from control lungs and neutrophils but was markedly increased in specimens from LPS-challenged rats. This increased NF-κB activity was inhibited by treatment with HES in a dose-related manner. The maximal inhibition of LPS-induced increase in NF-κB activity was observed at the HES dose of 7.5 mL/kg in both specimens. This was the same dose at which HES maximally inhibited an LPS-induced increase in lung microvascular permeability (Fig. 1), lung wet/dry weight ratio (Fig. 2), lung accumulation of neutrophils (Fig. 3), and lung CINC protein expression (Fig. 5).
The major focus of this study was to determine the effect of HES on lung capillary permeability in endotoxic rats and the mechanistic bases that underlie the possible effect. Our results showed that early treatment of HES (200/0.5) at doses of 3.75 and 7.5 mL/kg significantly reduced LPS-induced increases of lung capillary permeability.
Neutrophils play a central role in experimental models of acute lung leak. After being activated and mediated by adhesion molecules, integrins, and chemotactic factors, neutrophil accumulation is increased in lungs of endotoxic animal models. Neutrophils then release several toxic substances, including reactive oxygen species and proteolytic enzymes, that cause capillary leak and lung injury (9). In this respect, understanding the mechanisms by which HES reduces the increased capillary permeability should provide information on the regulation of processes that can contribute to the pathogenesis of acute lung leak. In this study, we provide evidence that HES could inhibit LPS-induced increases of lung neutrophil accumulation, CD11b expression on the blood neutrophil cell surface, lung CINC protein level, and NF-κB activation in both blood neutrophils and lungs in a dose-related manner. These findings provide the first in vivo data supporting a mechanism of HES-elicited reduction of capillary leak involving an antiinflammatory effect of HES, including inhibition of NF-κB activation.
Several studies have reported that HES molecules are able to reduce increases in microvascular permeability after ischemic insults in the spinal cord and cremaster muscle or after thermal burn (1,14,15). Our finding in an endotoxic rat model is consistent with these previous reports. In addition, our studies extend these previous observations by showing that the effect of HES is not dose dependent. Although doses of HES 3.75 and 7.5 mL/kg showed a significant reduction in lung capillary permeability, larger doses of 15 and 30 mL/kg did not. The question that arises is as follows: How do starch macromolecules affect the microvascular permeability? Zikria et al. (16) postulated that HES macromolecules act by physically sealing the barrier defects created by the injury. This hypothesis was formed on the basis that increased transport is associated with a widening of the interendothelial cleft in postcapillary venules. However, Suval et al. (17) found increased extravasation of macromolecules in the presence of normal microvascular ultrastructure. Furthermore, electron microscope evidence confirmed that separation of interendothelial clefts is not a necessary element for increases in microvascular permeability (18). Also, the dose-related manner of HES found here is hard to elucidate with only a physically sealing mechanism. There must be additional mechanistic bases that underlie the actions of HES. Oz et al. (14) demonstrated that HES might affect microvascular dysfunction by influencing neutrophil binding to stimulated endothelial cells. However, they did not know the mechanism of this effect. Pascual et al. (19) reported that HES could reduce neutrophil-mediated tissue injury through inhibition of neutrophil l-selectin expression. Our present findings suggest the in vivo linkage between improvement in microvascular permeability of HES and its reduction of neutrophilic inflammation, which involves its inhibition of CD11b expression, CINC level, and NF-κB activation, giving novel and general insight into the underlying mechanisms.
Activated neutrophils have been implicated as pathogenic mediators of injury to tissues and, in particular, to the microcirculation system. The critical common step is the adhesion of the activated neutrophils (5). Firm adhesion of neutrophils is mediated by β2 integrins (CD11b/CD18) and ICAM-1. The role of β2 integrins in lung injury has been determined by the application of blocking antibodies. Anti-CD11b antibodies were found to reduce complement-mediated lung injury (20). In vitro studies have tested the influence of HES on the expression of the adhesion molecule CD11b on neutrophils and found no significant inhibition effect of HES (21). However, the in vivo function has not been established. We observed that HES could inhibit LPS-induced increases of CD11b expression on neutrophils in a dose-dependent manner. This result is consistent with the hypothesis of Nohe et al. (22). They found that HES did not attenuate adhesion molecule expression but showed an immediate decreasing effect on neutrophil adhesion. They thought that this was due to its inhibition on interactions of neutrophilic β2 integrins with their endothelial counterreceptors.
CINC is structurally and functionally related to human IL-8. It is a potent chemotactic factor for rat neutrophils both in vitro and in vivo. Frevert et al. (23) reported that approximately 50% to 70% of neutrophil chemotaxis in LPS-induced pulmonary inflammation is caused by CINC. Because CINC itself can increase the expression of CD11b/CD18 integrin on rat neutrophils (23) and because expression of leukocyte adhesion molecules such as E selectin is dependent on CINC (24), the inhibition of lung CINC level by HES that we observed in our study may exert both direct and indirect effects on neutrophil vascular adhesion and extravascular migration.
In our study, we found that HES at doses of 3.75 and 7.5 mL/kg significantly reduced LPS-induced NF-κB activation in both blood neutrophils and lungs. NF-κB plays a central role in regulating the transcription of cytokines, adhesion molecules, and other mediators involved in acute respiratory distress syndrome, sepsis, and multiple organ system failure (10). Liu et al. (25) showed that inhibition of NF-κB activation in vivo suppresses LPS-induced CINC and ICAM expression, reduces neutrophil accumulation, and prevents capillary leak in multiple organs. It seems that HES exerts its actions through the NF-κB signaling pathway. We also found that HES could reduce the LPS-induced increase of TNF-α levels in plasma (data not shown). TNF-α is involved in NF-κB activation (26). It is possible that HES inhibits NF-κB activation by inhibition of TNF-α.
Emphasis should be placed on the hemodynamics, which are obviously important during the procedure because reversal of systemic hypotension would also influence microvascular disorders and thus would not allow discrimination between the effects of HES on macrohemodynamics and microcirculation per se. We measured the blood pressure of the rats in our pilot study, and this variable did not differ among the seven groups, indicating that the animals were hemodynamically stable. Minor hemodynamic changes probably could not be excluded because other measurements such as cardiac output (CO) and systemic vascular resistance (SVR) were lacking. We did not study CO and SVR in our experiment because, on the one hand, measurements of CO and SVR were invasive procedures and because repeated blood sampling of >1 mL during the procedure would likely influence the hemodynamics in such a limited circulating blood volume as in the rat; on the other hand, the changes of blood pressure were similar among the seven groups. However, these presumably minor changes would surely not influence hemodynamic stability. The fact that blood pressure differences among the groups were absent in the model used indicates that the improvements of microvascular permeability and reduction of neutrophilic inflammation by HES are not caused by reversal of macrohemodynamics disturbances.
Another noteworthy phenomenon in our experiment is that the maximal effect of HES was observed at a dose of 7.5 mL/kg in most cases. This may be because most mediators are regulated by many factors. Besides NF-κB, C/EBP and AP-1 are both important in the expression of cytokines, chemokines, and adhesive molecules. Maybe HES has some reverse effects on these pathways or on other untested mediators, such as selectins, and ICAM. Thus the effects of larger doses may be partially offset by those reverse effects. Further experiments are needed to confirm, in vivo, these and other proinflammatory signaling pathways on which HES might exert an influence. Finally, our experiments suggest the best dose of HES for use in a clinical setting. Because 2.5 mL in rats represents approximately 10% of the blood volume, 7.5 mL/kg is comparable to a human subject weighing 70 kg receiving 500 mL of HES.
In summary, we have shown that HES (200/0.5) at doses of 3.75 and 7.5 mL/kg significantly reduced LPS-induced increases in lung capillary permeability. The proposed mechanism is that HES inhibits LPS-induced NF-κB activation, followed by inhibition of the lung CINC protein level, CD11b expression on the blood neutrophil cell surface, and lung neutrophil accumulation, thus reducing the LPS-induced increase in microvascular endothelial permeability in the lung.
We thank Dr. Genbao Feng for technical assistance.
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