Bactericidal/Permeability-Increasing Protein Preserves Leukocyte Functions After Major Liver Resection

Abstract

Objective

To analyze postoperative leukocyte functions in patients undergoing hemihepatectomy, and to assess the effect of treatment with the endotoxin-neutralizing agent bactericidal/permeability-increasing protein (rBPI₂₁).

Summary Background Data

Extensive liver resection is associated with a high incidence of infectious complications. Because elimination of pathogenic microorganisms occurs mainly by leukocytes, this increased rate of infections is most likely due to an impaired function of these cells. Endotoxin, translocated from the gut into the systemic circulation as a result of increased gut permeability and reduced hepatic clearance function after major liver resection, may play an important role in the impairment of posthepatectomy leukocyte function.

Methods

To investigate whether hemihepatectomy results in impaired leukocyte functions and to determine the role of endotoxin in this process, leukocyte oxidative burst and leukocyte antigen expression were studied in three groups of patients: patients undergoing a hemihepatectomy and receiving rBPI₂₁ treatment, patients undergoing hemihepatectomy and receiving placebo, and as an extra control group patients undergoing other major abdominal surgeries. Blood samples were collected before surgery, 2 hours after surgery, and at days 1, 2, 5, and 7. Phorbol myristate acetate-stimulated oxidative burst was measured using dihydrorhodamine, and leukocyte surface expression of the antigens CD11b, CD16, and CD14 was investigated by indirect immunofluorescence. Both oxidative burst and membrane surface expression were quantified by flow cytometry. An indication of the antiendotoxin effect of rBPI₂₁ treatment was provided by assessment of plasma lipopolysaccharide binding protein (LBP) levels by enzyme-linked immunosorbent assay.

Results

The oxidative burst in the hemihepatectomized patients receiving placebo and the controls increased 2 hours after surgery, whereas it decreased in the rBPI₂₁-treated patients, resulting in significant differences between the groups. On day 1, neutrophil CD11b expression and monocyte CD14 expression in the rBPI₂₁-treated patients and controls were significantly lower than in the placebo group. At 2 hours, CD16 expression in the placebo-treated patients was significantly higher than in the rBPI₂₁-treated patients and controls. On day 5 and day 7, plasma LBP levels were significantly higher in the placebo-treated patients compared with the rBPI₂₁-treated patients.

Conclusions

The results of this study show that patients undergoing major liver resection have an increased activation of leukocytes compared with those undergoing other major abdominal surgery. This enhanced activation may contribute to the increased risk of infection in these patients. Administration of the endotoxin-neutralizing agent rBPI₂₁ to hemihepatectomy patients was shown to reduce plasma LBP levels, to preserve leukocyte functions partially, and to reduce leukocyte activation to the level of other, nonhepatic abdominal surgery.

Despite major advances in surgical techniques and improvements in perioperative care, extensive liver resection is still associated with a high incidence of infectious complications such as intraabdominal infections and sepsis. ^1–4 Because elimination of pathogenic microorganisms during the direct postoperative period occurs mainly by phagocytes such as polymorphonuclear leukocytes (PMNs) and monocytes, the development of postoperative infections may be due to an impaired function of these cells.

In recent years, postoperative defects were reported for all aspects of leukocyte function, including chemotaxis, ^5,6 phagocytosis, ^7,8 oxidative burst, ⁹ enzyme release, ¹⁰ and microbial killing. ¹¹ Further, postoperative release of proteolytic enzymes and production of reactive oxygen intermediates by these cells can lead to serious damage to endothelial cells and organs, as is seen during sepsis and multiple organ failure. ¹²

One of the potential mediators of impaired leukocyte function is endotoxin. After major liver resection, the hepatic mononuclear phagocytic system, the body’s main clearing mechanism for endotoxin, is reduced, ^13–16 allowing spillover of gut-derived bacteria and endotoxin into the systemic circulation. ^17,18 Indeed, endotoxin was demonstrated in plasma from patients undergoing a hemihepatectomy, whereas in plasma from patients undergoing other major abdominal surgery, endotoxin was not detectable. ¹⁹ Endotoxin can induce an enhanced expression of leukocyte and endothelial adhesion molecules and is a potent stimulator of the PMN oxidative burst in vitro and in vivo. ^20–22 Therefore, an endotoxin-induced change of PMN function could provide an explanation for the increased occurrence of infections and sepsislike complications after major liver surgery.

The goals of the present study were twofold. First, we examined whether major liver surgery (i.e., resection of a large part of the liver parenchyma) is associated with an enhanced activation of leukocytes. To test this hypothesis, we compared the effects of hemihepatectomy with those of other major abdominal surgery on functional (oxidative burst capacity) and important phenotypic leukocyte markers (expression of leukocyte receptors CD11b, CD16, CD14). Second, we indirectly tested the hypothesis that endotoxin plays a prominent role in leukocyte activation after major liver resection by using rBPI₂₁, a 21-kD recombinant amino-terminal protein derived from the 55 kD-human bactericidal/permeability-increasing protein (BPI), which is normally found in azurophilic granules of human neutrophils. Like human BPI, rBPI₂₁ is antibacterial and binds to circulating lipopolysaccharide on or derived from gram-negative bacteria. As a result of this binding, rBPI₂₁ not only clears endotoxin but also inhibits several endotoxin-induced humoral and cellular responses, including the generation of cytokines, free radicals, or nitric oxide, induction of tissue factor, adhesion of neutrophils to endothelial cells, and complement activation. ^23–29 By treating the hemihepatectomy patients with this endotoxin-neutralizing agent rBPI₂₁, we could estimate the effect of systemic endotoxemia in leukocyte activation after liver surgery. The effectiveness of rBPI₂₁ treatment was measured by assessing plasma lipopolysaccharide binding protein (LBP) levels.

METHODS

The study was approved by the Council for Medical Research of the Netherlands Organization for Scientific Research and by the review board of our institution. Informed consent was obtained from all patients.

Patients

The hemihepatectomy patients in this study were part of a phase II, double-blind, placebo-controlled, multicenter, dose-escalation trial with rBPI₂₁ in the prevention of infectious complications after liver resection. Patient inclusion was primarily based on performance of a resection of three or more liver segments. The two hemihepatectomy groups (placebo and rBPI₂₁) were compared with a group of patients undergoing major abdominal resection for nonhepatic abdominal tumors (controls). APACHE III scores were assessed before surgery and at 2 hours and 1, 2, and 7 days after surgery. Infectious events were classified by the usual clinical criteria and recorded during the first 14 postoperative days.

The hemihepatectomy patients were randomly assigned in a 1:1 ratio to treatment with either 8 mg/kg/48 hours rBPI₂₁ or placebo in an equal volume by a 48-hour continuous intravenous infusion, starting about 1 hour before resection of the liver parenchyma. Patients, investigators, and clinical monitors remained unaware of individual treatment until all data were processed and statistically evaluated.

rBPI₂₁ (Neuprex) was supplied by XOMA Corp. (Berkeley, CA) in an aqueous buffer plus stabilizers as a clear, colorless, sterile nonpyrogenic solution. A matching placebo was also supplied as a clear, colorless, pyrogen-free solution in aqueous buffer without stabilizers. Throughout the dosing procedure, standard aseptic technique for intravenous administration was used. None of the patients had other diseases or conditions causing immunosuppression, nor did they receive immunosuppressive therapy during the perioperative period. In addition to study medication, all patients received conventional supportive therapy according to good clinical practice.

Blood Samples and Preparation of Leukocytes

Peripheral heparinized blood samples were collected before surgery (baseline) and at 2 hours and 1, 2, and 7 days after surgery. The samples were processed within 1 hour. Three milliliters of blood was layered over 3 mL Lymphoprep (Nycomed Pharma AS, Oslo, Norway) and was allowed to sediment by gravity for 40 minutes at room temperature. Leukocytes were harvested and pelleted by centrifugation (300 g for 5 minutes at 4°C). Contaminating erythrocytes in the pellet were lysed by 5 minutes of incubation in an ice-cold isotonic solution of 0.83% (w/v) NH₄Cl and 0.084% (w/v) NaHCO₃, after which cells were centrifuged again. The leukocyte-containing pellet was washed twice with calcium- and magnesium-free phosphate-buffered saline containing 0.5% (w/v) bovine serum albumin (PBS-BSA). Finally, the cells were counted and resuspended in PBS-BSA at a concentration of 2.5 × 10⁶ cells per milliliter and were kept at 4°C in the dark. The number of nonviable cells, determined by staining with propidium iodide, was negligible (<2%).

PMN Oxidative Burst Activity

The production of oxygen radicals was measured using the well-established method of Rothe et al. ^30,31 Briefly, 20 μL of a 10-ng/mL phorbol myristate acetate (PMA) solution was added to polystyrene round-bottom tubes containing 200 μL leukocyte suspension and 10 μL of a 10-μmol/L solution of dihydrorhodamine (DHR). The mixtures were incubated in a water bath for 20 minutes at 37°C, after which stimulation was stopped by addition of 400 μL ice-cold PBS-BSA. Cells were kept at 4°C in the dark until the oxidative burst was quantified by flow cytometry.

Phenotypic Analysis of PMNs and Monocytes

PMN membrane surface expression of CD11b and CD16 and monocyte surface expression of CD14 were investigated by indirect immunofluorescence. For each assay, 2.5 μL specific monoclonal mouse antibodies was added to 25 μL heparinized whole blood. The antibodies used were CLB-B2.12 (CD11b), CLB-FcR-gran1 (CD16) (CLB, Amsterdam, The Netherlands), and anti-CD14 (Becton Dickinson Immunocytometry Systems, San Jose, CA). The mixtures were then incubated on ice for 30 minutes in the dark, after which cells were centrifuged (300 g for 5 minutes at 4°C) and washed three times with PBS-BSA. Labeling was performed with 25 μL phycoerythrin-labeled rabbit antimouse antibodies (Dakopatts A/S, Glostrup, Denmark) for 30 minutes on ice in the dark. Erythrocytes were lysed, and the remaining white blood cells were washed again. After addition of 200 μL PBS-BSA, cells were kept at 4°C in the dark until antigen expression was quantified by flow cytometry as mean cellular fluorescence.

Flow Cytometry

Flow cytometric analysis of cell-associated fluorescence was performed on a FACScan (Becton Dickinson, Mountain View, CA). Polymorphonuclear leukocytes and monocytes were recognized by their characteristic pattern of light scatter and could easily be discriminated from lymphocytes, thrombocytes, erythrocytes, and cell fragments. By setting appropriate gates, PMN and monocyte populations were analyzed without further purification. Cell surface antigen expression and oxidative burst were assessed by measuring the red fluorescence of, respectively, phycoerythrin and DHR. Each time, 10,000 cells were analyzed per tube, and fluorescence distribution was displayed as a single histogram. The events were acquired in a linear mode for forward scatter and side scatter and in logarithmic mode for red fluorescence. Antigen expression and oxidative burst were expressed as the mean fluorescence of all gated cells.

Analysis of Plasma LBP Levels

Plasma samples were obtained before surgery (baseline) and at 1, 2, 3, 5, and 7 days after surgery using endotoxin-free heparinized blood samples. Blood samples were placed on ice and then centrifuged at 4°C for 15 minutes at 1500 g within 30 minutes. Tubes were then frozen at −70°C and shipped on dry ice to a single research laboratory. Plasma LBP determinations were done by enzyme-linked immunosorbent assay, as described elsewhere. ³² For detection of LBP, plates were coated with polyclonal antihuman LBP antibodies. Diluted plasma samples and recombinant LBP standard dilution series were added to the plate. Detection occurred with a biotinylated polyclonal rabbit antihuman LBP IgG, followed by peroxidase-conjugated streptavidin and TMB (KPL, Gaithersburg, MD). The detection limit was 500 pg/mL.

Statistical Analysis

Data were analyzed with SPSS 7.5 (SPSS, Inc., Chicago, IL). All results are expressed as mean ± standard error of the mean. Overall differences between groups were analyzed by two-way analysis of variance; if a significant overall difference between groups was found, differences between groups at predefined time points were analyzed using the two-sample Mann-Whitney test. Differences within groups were analyzed with the Wilcoxon rank test for two related samples. Significance was accepted at a two-tailed P < .05.

RESULTS

Demographics

A total of 22 patients was included in this study: 12 patients undergoing a hemihepatectomy for liver metastasis of colorectal carcinoma and 10 patients undergoing major abdominal surgery for nonhepatic malignancies. Four of the six hemihepatectomy patients who received rBPI₂₁ underwent a right hepatic lobectomy (resection of segments 5–8), one patient underwent an extended right hepatic lobectomy (segments 4–8), and one patient underwent an extended left hepatic lobectomy (segments 2–4). The resections in the six placebo-treated hemihepatectomy patients were comparable with those of the rBPI₂₁ group and consisted of three right hepatic lobectomies, two extended right hepatic lobectomies, and one extended left hepatic lobectomy. In the control group, the operations were three right hemicolectomies, three rectum and sigmoid resections, two distal esophagus and stomach resections, and two pancreaticoduodenectomies (Whipple procedure). Mean age, distribution of sexes, mean time of operation, and blood loss did not differ among the three groups (Table 1).

Table 1. DEMOGRAPHICS AND OPERATIVE DATA

Data are expressed as mean ± SEM.

Clinical Parameters

APACHE III scores were significantly increased 2 hours after surgery in all groups, indicating the extent of surgical trauma (Fig. 1). Control patients had a lower score at day 1 and 2, but this difference was not significant (day 1, P = .09). Although evaluation of the clinical course was not a main purpose of this study, the presence of infectious complications in the three groups was recorded daily. Four of the six placebo-treated hemihepatectomy patients (67%) developed one or more infectious complications (sepsis, n = 2; pneumonia, n = 2; and one case each of bacteremia, intraabdominal infection, wound infection, urinary tract infection). In the rBPI₂₁ group, two patients (33%) had infectious complications (sepsis and intraabdominal infection). Only 2 of the 10 control patients (20%) had infectious complications; both patients were septic as a result of pneumonia.

Figure 1. Course of the APACHE III scores from the control patients (□), the placebo-treated hemihepatectomy patients (○), and the rBPI₂₁-treated hemihepatectomy patients (•). There were no significant differences among the groups. Data are expressed as means ± standard error of the mean.

Oxidative Burst Activity

PMN oxygen radical production in response to PMA stimulation was measured as an indication of leukocyte activation (Fig. 2). Before surgery, the oxidative burst was similar in all groups. After surgery, the oxidative burst of the hemihepatectomized patients receiving placebo and the controls showed an increase, whereas the oxygen radical production in the rBPI₂₁ group decreased, resulting in a significant difference among all groups at 2 hours after surgery (P = .03). There was also a significant difference between the placebo group and the rBPI₂₁ group (P = .03).

Figure 2. Phorbol myristate acetate-stimulated oxidative burst activity as measured by mean channel fluorescence before and during the first week after surgery by the polymorphonuclear leukocytes of control patients (□), placebo-treated hemihepatectomy patients (○), and rBPI₂₁-treated hemihepatectomy patients (•). Significant differences between the groups are indicated by asterisks. Data are expressed as means ± standard error of the mean.

PMN CD11b Receptor Expression

The expression of CD11b, a member of the β2 integrin adhesion molecule family, is shown in Figure 3A. Before surgery and at 2 hours after surgery, CD11b expression was similar in all groups. However, on day 1, CD11b expression in the rBPI₂₁ group and controls was decreased, whereas in the placebo group there was only a moderate decline, resulting in significant differences between the placebo group and the control group (P = .04) and the placebo group and the rBPI₂₁ group (P = .04). Differences between groups disappeared on day 2 and day 7.

Figure 3. Expression of CD11b (A) and CD16 (B) was measured by mean channel fluorescence before and during the first week after surgery on polymorphonuclear leukocytes in control patients (□), placebo-treated hemihepatectomy patients (○), and rBPI₂₁-treated hemihepatectomy patients (•). Significant differences between the groups are indicated by asterisks. Data are expressed as means ± standard error of the mean.

PMN FcγRIII (CD16) Receptor Expression

The expression of the IgG receptor FcγRIII (CD16) was measured to assess the activation state of PMNs (Fig. 3B). CD16 receptor expression showed an almost similar trend to that of the oxidative burst, with an increased expression at 2 hours in the placebo group and decreased expression in the rBPI₂₁ group. CD16 receptor expression in the controls remained stable. The difference between the placebo group and the control group (P = .002) was highly significant, indicating the difference in leukocyte activation after liver resection and nonhepatic abdominal surgery. Notably, this difference was not found between the rBPI₂₁ group and the controls. From day 1 onward, CD16 expression was comparable among the groups.

Monocyte CD14 Receptor Expression

Monocyte activation was measured by the assessment of CD14 receptor expression (Fig. 4). At 2 hours, all groups showed an increased expression of monocyte CD14, which was followed by a decrease during the next days. This decrease was slower in the placebo-treated hemihepatectomized patients than in the rBPI₂₁-treated patients and the controls. At day 1, CD14 expression in the placebo group was significantly higher than in the controls (P = .01), but there was no difference between the rBPI₂₁ group and the controls.

Figure 4. Perioperative CD14 expression on monocytes of control patients (□), placebo-treated hemihepatectomy patients (○), and rBPI₂₁-treated hemihepatectomy patients (•), measured by mean channel fluorescence. Significant differences between the groups are indicated by asterisks. Data are expressed as means ± standard error of the mean.

Plasma LBP Levels in Hemihepatectomy Patients

Plasma LBP levels were measured in the two hemihepatectomy groups as an indication of the antiendotoxin effect of rBPI₂₁ treatment (Fig. 5). Before surgery, the plasma levels of LBP in the placebo-treated and rBPI₂₁-treated patients were identical and similar to the levels found in healthy volunteers (<7.4 μL/mL), but during the first postoperative days plasma LBP levels increased. At day 5 and 7, the levels declined in the rBPI₂₁-treated patients, but in the placebo-treated patients the levels still increased, resulting in a significant difference between the groups at day 5 (P = .04) and 7 (P = .03).

Figure 5. Perioperative plasma lipopolysaccharide binding protein levels in placebo-treated hemihepatectomy patients (○) and rBPI₂₁-treated hemihepatectomy patients (•), measured by enzyme-linked immunosorbent assay. Significant differences between the groups are indicated by asterisks. Data are expressed as means ± standard error of the mean.

DISCUSSION

Partial hepatectomy, the first choice of treatment for patients with liver metastasis of colorectal cancer, remains associated with a high incidence of infectious complications. The occurrence of these infectious complications in the immediate postoperative period is most likely due to a phagocytic cell dysfunction. Major surgery affects leukocyte functions, and changes have been reported in chemotaxis, enzyme release, microbial killing, and oxidative burst. 5,6,9–11 Previously, our group has reported a decreased leukocyte phagocytic capacity in surgical patients with liver metastases. ⁸ After liver resection, this phagocytic capacity was still further decreased, but such a decrease was also found in patients undergoing major abdominal surgery for nonhepatic malignancies. However, in this study the postoperative impaired phagocytosis could be explained by decreased levels of the plasma opsonins C3a and IgG, and it therefore was probably not a result of an impaired leukocyte function. Apart from reports of decreased leukocyte functions after surgery, there is evidence that enhanced activation of leukocytes is also an important factor in the development of serious complications. ^33,34

In keeping with the results of others, the present data showed that PMN oxygen radical production was increased just after major abdominal surgery. We also found that in the placebo-treated hemihepatectomized patients, the increase was even more pronounced. However, treatment of hemihepatectomy patients with rBPI₂₁ resulted in a reduction of the oxidative burst to levels below that of the controls. Notably, these differences occurred despite comparable baseline values in all groups and a similar APACHE III score before and just after surgery.

The important role of reactive oxygen intermediates in the pathogenesis of sepsis is well accepted. ¹² It was shown that an early postoperative increase in oxygen radical production correlates with subsequent development of sepsis. ³⁵ Further, a significantly higher oxidative burst was found in surgical patients with postoperative infections compared with patients with an uncomplicated postoperative course. ^36,37 Therefore, our experimental data seem to confirm that liver surgery is associated with a higher risk of septic complications compared with other abdominal surgery for malignant diseases. The data further suggest that treatment of patients undergoing a hemihepatectomy with the endotoxin-neutralizing rBPI₂₁ may be beneficial by decreasing PMN hyperactivity and oxygen radical production during the early postoperative period, thereby reducing the risk of enhanced tissue damage and subsequent complications.

The level of expression of PMN CD11b, an essential receptor for the process of extravasation and migration to the site of infection, has been proposed as a useful marker for neutrophil activation in clinical studies. ³⁸ Further, it is well established that major surgery is accompanied by increased expression of leukocyte CD11b, and recent literature has provided evidence that increased CD11b expression on the first postoperative day can predict the development of sepsis. ³⁵ Especially in ischemia-reperfusion injury after major vascular surgery, such as thoracoabdominal aortic aneurysm repair, early CD11b expression is a useful predictor of outcome. ³⁹ We found that neutrophil CD11b expression was markedly increased in the controls just after major abdominal surgery and subsequently decreased on day 1. Hemihepatectomized patients receiving placebo initially showed a similar course of CD11b expression, but it stayed at a higher level and subsequently decreased more slowly. In contrast, the rBPI₂₁-treated patients showed a postoperative course of CD11b expression that was similar to that of the controls, indicating that treatment with rBPI₂₁ could reduce leukocyte activation after hemihepatectomy to the level of nonhepatic abdominal surgery. Even though the functional significance of this downregulation remains unclear, the established correlation with better outcome does suggest a favorable action of rBPI₂₁ in major liver surgery.

Another important cell surface marker on leukocytes is CD16, the receptor for the Fcγ region of IgG. Cross-linking of this receptor induces activation of the oxidative burst, and in patients undergoing major resectional surgery it was shown that patients with postoperative sepsis had a constitutively higher expression of neutrophil CD16 both before and after surgery. ⁴⁰ In the control group, we found almost no effect of surgery on the level of surface neutrophil CD16 expression, confirming the results of others. ⁴⁰ However, in the placebo-treated hemihepatectomy patients, we found an increased expression just after surgery. Again, treatment with rBPI₂₁ prevented this increase in the hemihepatectomy patients.

We also measured the monocyte CD14 receptor, a cell surface antigen that is the main endotoxin receptor on leukocytes. ⁴¹ Monocyte CD14 can be released by cells to yield a soluble protein (sCD14) and therefore exists in both membrane-associated and soluble forms. Elevated plasma concentrations of sCD14, together with a reduced expression of mCD14, have been reported in patients with pathologic conditions such as sepsis, infection, or multiple organ failure, and increased serum levels of sCD14 were associated with a higher death rate. ^42,43 In the control patients and the rBPI₂₁-treated patients, the initial rise of CD14 expression at 2 hours after surgery was followed by a fast decrease, a pattern similar to that seen in patients undergoing cardiopulmonary bypass. ⁴⁴ However, in the placebo-treated hemihepatectomy patients, this decrease was much slower, resulting in significantly higher CD14 expression on day 1.

The present study was not designed to correlate postoperative PMN functions with clinical outcome, but our clinical data agree with the results of the oxidative burst and antigen expression assays. Four of the six placebo-treated hemihepatectomy patients (67%) had one or more infectious complications, compared with only two of the six rBPI₂₁-treated patients (33%). Even though the difference was not significant, the longer mean surgical time and the higher mean blood loss in the rBPI₂₁-treated patients make these data even more notable. At only 2 of 12 patients (17%), the rate of infectious complications in the control group was even lower. However, this study was not conducted to prove the clinical effects of rBPI₂₁ treatment, and the small number of patients does not allow us to draw any conclusions in terms of outcome.

With the administration of rBPI₂₁, we indirectly sought to investigate the role of endotoxin on neutrophil function in hemihepatectomy patients. The most direct proof that rBPI₂₁ treatment is effective and results in decreased plasma levels of endotoxin would be direct assessment of plasma endotoxin levels in rBPI₂₁-treated and placebo-treated patients. However, measurement of endotoxin in the systemic circulation is difficult. The commonly used Limulus amebocyte lysate reactivity assay is susceptible to several interfering substances found in human plasma that may activate or inhibit the limulus reaction independent of endotoxin itself. ^45,46 Thus, although some studies have shown that endotoxin measurements may be of value in clinical research, others have been inconclusive, and the clinical value of endotoxin measurements in patients remains unclear. ^47–50 In this study, we anticipated this difficulty and therefore chose to assess systemic endotoxemia indirectly by use of the more reliable LBP, the principal plasma protein responsible for transporting endotoxin to the immune effector cells. ⁵¹ LBP levels have been shown to rise in healthy persons injected with endotoxin, and longitudinal sampling from patients undergoing liver surgery or patients with hemorrhage resulting from trauma also shows elevated LBP levels. Indeed, the placebo-treated patients showed significantly higher plasma LBP levels compared with the rBPI₂₁-treated patients, strongly suggesting the effectiveness of the endotoxin-neutralizing rBPI₂₁ treatment.

Endotoxin is a potent stimulant of leukocyte activation, both by direct cellular stimulation and indirectly by initiation of the acute-phase cytokine production by monocytes. In vitro experiments have shown that endotoxin exposure results in increased leukocyte adhesion receptor expression and enhanced oxidative radical production. ^19–21,52 Evidence for the in vivo role of endotoxin in leukocyte activation comes from studies in patients undergoing thoracoabdominal aortic aneurysm repair. ³⁹ In these patients, a highly significant correlation was found between leukocyte CD11b expression and plasma endotoxin levels. Animal studies have shown that exposure of the peritoneal cavity to air is a potent stimulator of gut-derived endotoxin translocation across the gut. ⁵³ According to that theory, the three groups were equally at risk for endotoxin translocation from the gut because they had a similar period of exposure to air. However, because of the reduction of the mononuclear phagocytic system in the hemihepatectomized patients, the chance of subsequent endotoxemia was much greater in these patients. This may explain the higher initial oxidative burst and antigen expression in the placebo-treated hemihepatectomy patients and the reduction of these functional parameters in the rBPI₂₁-treated hemihepatectomy patients. By neutralizing endotoxin, the systemic endotoxemia may have been prevented in the rBPI₂₁-treated hemihepatectomy patients, resulting in a reduction of the activation state of leukocytes to the level of the controls. However, the rBPI₂₁-induced changes in neutrophil activation may also have another biologic basis.

In conclusion, major liver resection is associated with an enhanced early postoperative leukocyte activation, as expressed by the higher oxidative burst and leukocyte surface marker expression. Treatment with rBPI₂₁ can reduce leukocyte activation in hemihepatectomy patients to the level of other, nonhepatic major abdominal surgery and may therefore be a potential agent in the prevention of tissue damage and septic complications after major liver surgery.

Acknowledgments

The authors thank Petra Teiwes for her valuable assistance during the study and Alexander Houdijk and Geertrui Vanhove for their careful review of the manuscript.