INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The acute respiratory distress syndrome (ARDS) is characterized by damage to the pulmonary capillary endothelium and epithelium, and an intense inflammatory process in the lung parenchyma (1). This form of acute lung injury, which is encountered most commonly in sepsis, can also involve dysfunction of other organs, including kidney, liver, gut and heart. Activation of vascular endothelium and recruitment of activated leukocytes contributes to injury in ARDS and multiple-system organ dysfunction (2). During inflammation, cellular adhesion molecules are upregulated and promote leukocyte adherence to endothelium by attachment to specific ligands (2). One important family of adhesion molecules, the selectins, mediates the initial loose interaction between leukocytes and endothelium, often referred to as "rolling" (2). This loose adhesion occurs prior to tight adhesion and migration of leukocytes into tissue, which are mediated by the integrins and the molecules of the immunoglobulin supergene family (2). Polymorphonuclear leukocytes (PMN) have been implicated in the pathogenesis of acute lung injury in sepsis, and are present in increased number in both lung parenchyma and bronchoalveolar lavage fluid (BALF) of patients with ARDS (3, 4). Furthermore, neutrophil depletion or blockade or influx of lung PMN decreases organ injury in several types of experimental inflammatory lung injury (1).

The three known selectins, E-, P-, and L-selectin, are glycoproteins of similar structure comprising a lectin domain, an epidermal growth factor(EGF)-like domain, variable repeating numbers of complement regulatory protein-like regions, a transmembrane domain, and a cytoplasmic tail (5). These molecules have independent induction but overlapping function in leukocyte adherence to vascular endothelium (6). After exposure to inflammatory mediators, E-selectin is induced within 6 h on the surface of endothelial cells, where it initiates PMN binding (5). E-selectin is upregulated during systemic inflammatory responses such as immune-complex lung injury in rats (8), E. coli infusion in baboons (9, 10), and lipopolysaccharide (LPS) infusion in monkeys (11). L-selectin is constitutively expressed on leukocytes, including lymphocytes, PMN, and monocytes, and participates in neutrophil and monocyte adhesion to activated vascular endothelium in vitro and in vivo (12).

Blocking antibodies for E-selectin and L-selectin in experimental models of inflammation have decreased tissue neutrophil accumulation, vascular permeability, and organ injury (8, 12). Additionally, studies of immune-complex lung injury in rats showed that a combination of antibodies to E- and L-selectin was more effective in preventing neutrophil accumulation and lung injury than either antibody alone (16). These findings support the hypothesis that selectins play a pivotal role in PMN recruitment into tissues in many pathologic states. Since sepsis-induced acute lung injury is associated with recruitment of PMN, these results provide a rationale for single and dual blockade of selectins to prevent lung injury in sepsis. EL-246 is a novel antibody with immunologic and functional blocking activity against human E- and L-selectin (17). This antihuman monoclonal antibody (mAb) cross reacts with tissues of nonhuman primates as well as other species (18), and in preliminary flow-cytometric studies reacted with leukocytes from the species Papio cynocephalus, used in this study. This antibody has been reported to decrease acute lung injury in septic pigs (18) and to increase survival in sheep after ischemia-reperfusion lung injury (19).

Our laboratory has used E. coli sepsis to produce acute lung injury and multiple-organ dysfunction in baboons. Like human sepsis, this injury is characterized by increased PMN and severe injury of the lung and other organs (20). We hypothesized that pretreatment of baboons with EL-246 prior to a lethal dose of E. coli would attenuate PMN influx into the tissues, result in less organ injury, and prolong survival time.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Materials

Unless otherwise specified, all chemicals were purchased from Sigma Chemical Co., St. Louis, MO.

Animal Model

Fourteen adult male baboons (P. cynocephalus) weighing 14 to 20 kg were used for these studies. Colony-bred animals were obtained from the Southwest Foundation for Biomedical Research in San Antonio, TX, and were admitted to the Duke Vivarium, where they were quarantined for a minimum of 4 wk. They were confirmed to be negative for tuberculosis by skin test prior to use. The animals were handled in accordance with American Association for the Accreditation of Laboratory Animal Care guidelines, using a protocol approved by the Duke University Institutional Review Board and Institutional Animal Care and Use Committee. They were fasted overnight prior to the start of the experiment.

The baboons were sedated initially with intramuscular ketamine (20 to 25 mg/kg; Parke-Davis, Inc., Morris Plains, NJ) and then intubated with a size 6 endotracheal tube. A saphenous vein catheter was placed for infusion of medications. A femoral incision was made, using sterile technique, and an arterial catheter was inserted for blood-pressure monitoring and sampling of arterial blood. At this site, a size 5-French pulmonary artery catheter was inserted to measure central venous, pulmonary artery, and pulmonary capillary wedge pressures, to collect mixed venous blood, and to determine cardiac output (CO).

The animals were ventilated with a volume-cycled ventilator (Bear 2; Bear Medical Systems, Riverside, CA) with 21% oxygen at tidal volumes of 10 to 12 ml/kg, a positive end-expiratory pressure of 2.5 cm H₂O, and a respiratory rate sufficient to maintain an arterial PCO₂ of 35 to 40 and pH in the range of 7.40 ± 0.05. Sedation was maintained with ketamine (3 to 10 mg/kg) and diazepam (0.4 to 0.8 mg/kg) every 2 h. The animals were paralyzed intermittently with pancuronium (0.1 mg/kg; Gensia Pharmaceuticals, Inc., New York, NY) to obtain physiologic measurements. To prevent iatrogenic infections and ventilator-associated pneumonia, ampicillin 1 g intravenously was administered every 6 h (Marsam Pharmaceuticals, Inc., Cherry Hill, NJ). Polymyxin B (20,000 U; Pfizer Inc., New York, NY) and gentamicin (40 mg; Solopak Laboratories Inc., Elk Grove Village, IL) were given intratracheally every 2 h, in conjunction with oral and endotracheal suctioning.

Experimental Protocol

Twelve of the 14 baboons used for this study were allocated into two groups of six animals each to compare a group of septic control animals with a group of septic animals that received EL-246 antibody (18) prior to sepsis. Six baboons received 1 mg/kg of EL-246 intravenously prior to infusion of live E. coli (serotype 086a:k61; American Type Culture Collection, Rockville, MD). Six baboons received the E. coli infusion without the antibody to serve as the control group. Two other animals received antibody infusion without E. coli to provide controls for the antibody therapy.

The protocol for infusion of E. coli was identical in the two experimental sepsis groups. The baboons received a sublethal dose of heat-killed (HK) E. coli (1 × 10⁹ cfu/kg) intravenously at the beginning of the experiment (t = 0). Twelve hours later (t = 12), a lethal dose of live E. coli (1 × 10¹⁰ cfu/kg) was administered intravenously over a period of 1 h. Two hours after the start of the live E. coli infusion, the animals received gentamicin (3 mg/kg intravenously). The antibody-treated group received EL-246 (1 mg/kg) immediately before the infusion of live bacteria (t = 12).

After preparation, the animals were observed for 6 h for hemodynamic stability before the experiment was begun. During this time, baseline hemodynamic, hematologic, respiratory, and A/ measurements were made. A maintenance intravenous infusion of lactated Ringer's solution (Abbott Laboratories, North Chicago, IL) was administered at 30 ml/h. The infusion rate was increased as needed during the experiments to maintain a mean arterial pressure (MAP) of 60 mm Hg and a pulmonary capillary wedge pressure (PCWP) of 8 to 12 cm H₂O. Temperature (T), heart rate (HR), mean arterial blood pressure (MABP), pulmonary artery pressures (Ppa), ventilator parameters, and fluid balance were recorded every hour. Every 6 h, PCWP, CO, and arterial and mixed venous blood gas tensions were obtained. Oxygen delivery (DO₂) and systemic and pulmonary vascular resistances (SVR, PVR) were calculated from these measurements and normalized to body weight. Complete blood counts were performed every 12 h (Symex-1000 Hemocytometer; Symex, Inc., Long Grove, IL), beginning just before the initial infusion of HK bacteria (t = 0). Additional blood counts were done at 1 h and 6 h after infusion of live bacteria (t = 13 and t = 18), respectively. Serum from these blood specimens was stored at 80° C for later quantitation of EL-246 levels. A/ measurements were made at baseline and at 12-h, 18-h, 36-h, and 42-h time points.

The animals were maintained according to the experimental protocol until death or for 36 h after infusion of live E. coli (t = 48). Criteria for early termination were hypotension or metabolic acidosis refractory to intravenous fluids and dopamine, or hypoxemia requiring inspired oxygen concentrations above 40%. The fraction of inspired oxygen (FI_O2) was limited to prevent pulmonary oxygen toxicity, which would confound interpretation of the extent of lung injury. Animals were killed at the end of the experiments by intravenous injection of saturated KCl solution, after the induction of deep anesthesia. Immediate autopsy was performed, with harvest and preparation of tissues as subsequently described.

Tissue Harvest and Preparation

After death, the thorax of each animal was opened and the left lung was removed after ligation of the left mainstem bronchus. The right lung was inflation-fixed for 15 min in situ via the endotracheal tube with 2% glutaraldehyde in 0.85 M sodium cacodylate buffer (pH 7.4) at a pressure of 30 cm H₂O. The right lung was then removed en bloc and immersed in this fixative. Sections were later embedded into paraffin or processed for electron microscopy.

Bronchoalveolar lavage (BAL) was performed on the left upper lobe immediately after removal of the left lung, using 240 ml 0.9% saline. Samples were frozen at 80° C for later analysis of protein and lactate dehydrogenase (LDH) content. Tissue from the heart, liver, kidney, small bowel, and lung were snap frozen in liquid nitrogen and stored at 80° C for later quantitation of myeloperoxidase (MPO) and thiobarbituric acid-reactive substances (TBARS), and for Western blot assay.

Quantitation of Ventilation-Perfusion Relationships

A/ distributions were measured with the multiple inert gas elimination technique described by Wagner and colleagues (21). Measurements were made at 0 h, 12 h, 18 h, 36 h, and 42 h. Six inert gases (sulfur hexafluoride, ethane, cyclopropane, enflurane, diethyl ether, and acetone) were mixed in normal saline and infused continuously via a peripheral vein at a rate of 2.5 to 3 ml/min. Samples of arterial blood, mixed venous blood, and mixed expired gases were collected in duplicate at 40 min after the beginning of the infusion. Each sample was equilibrated with an equal volume of nitrogen in a heated water bath at 37 ° C for 40 min. The expired gas samples and equilibrated blood samples were analyzed for sulfur hexafluoride with gas-liquid chromatography (GLC), using an electron capture detector, and for the other five gases with GLC and a flame ionization detector (Gas Chromatograph, Model 5890, Series II; Hewlett-Packard, Inc.). The data were directed to an analog-to-digital converter and stored on a computer for analysis. The blood-gas partition coefficients of the six inert gases were determined each day. Retention-partition coefficient and excretion-partition coefficient curves were generated through the techniques of enforced smoothing and least-squares analysis. The data were then transformed into A/ distribution, using a 50-compartment lung model. Shunt was defined as a A/ < 0.005, and dead space as a A/ > 100. The log standard deviations of perfusion and ventilation (SDq and SDv) were used as indices of blood flow and ventilation, respectively.

Biochemical Measurements

EL-246 measurements. Levels of EL-246 in the serum of four animals were quantitated with an enzyme-linked immunosorbent assay (ELISA).

MPO assay. The MPO assay was performed as described by Kensler and associates, with modification (22). Tissues were weighed and homogenized, mixed with an equal volume of 1% hexadecyltrimethyl ammonium bromide in phosphate buffer (pH 6.0), and rehomogenized. The suspension was freeze-thawed three times in liquid nitrogen, rehomogenized, and centrifuged at 15,000 × g for 5 min at 4° C. The supernatant was added to phosphate buffer (50 mM, pH 6.0), containing o-dianisidine (8.4 mg/ml) and 0.0005% H₂O₂, and the resulting solution was analyzed in a spectrophotometer (U-2000; Hitachi Instruments, Inc., Norcross, GA) for change in absorbance at 460 nm over a period of 3 min. MPO activity was expressed as change in absorbance/min/g wet-weight tissue.

Thiobarbituric acid-reactive substances assay. The supernatants prepared for MPO assay were used in the TBARS assay. Aliquots of supernatant were mixed with equal volumes of 3.5% sodium dodecyl sulfate (SDS; Bio-Rad, Hercules, CA) and 0.13 ml of 100% acetic acid, and the pH was adjusted to 3.5. Five milliliters of 0.6% thiobarbituric acid was added to each tube, and the tubes were heated in an oil bath at 95° C for 30 min. After cooling, 1.5 ml of n-butanol:pyridine (15:1) was added, and the tubes were vortexed for 30 s and centrifuged at 4,000 rpm for 10 min. The absorbance values of the supernatants were measured at 525 nm. Values were reported as absorbance per gram of wet weight of tissue.

Protein and LDH. The protein concentration of serum samples, BALF, and tissue homogenates was measured with the bicinchoninic acid assay, using bovine serum albumin (BSA) as a standard (23). LDH was measured by conversion of L-lactate and nicotinamide adenine dinucleotide (NAD⁺) to pyruvate and NADH. The BALF was mixed with 6.5 mmol/L NAD⁺, 52 mmol/L L-lactate, 55 mmol/L KCl, and 100 mmol/L Tris. Spectrophotometric absorbance was measured at 339 nm over a period from 0 s to 180 s. The values were expressed in units of activity per liter (U/L).

Western Blotting

Serum samples obtained at multiple time points from two sepsis control animals were mixed with cold lysis buffer (150 mM NaCl, 50 mM Tris, pH 7.6, 1% SDS, 3% Nonidet P-40, 5 mM ethylene diamine tetraacetic acid (EDTA), 1 mM MgCl₂, 2 mM 1,3-dichloroisocoumarin, 2 mM 1,10-phenanthroline, and 0.4 mM E-64). The serum was mixed with an equal volume of Laemmli sample buffer (250 mM Tris-HCl, pH 6.8, 4% SDS, 10% glycerol, 0.006% bromphenol blue, and 2% -mercaptoethanol) and was stored at 80° C until used. Lung samples from each group plus two historical controls were homogenized in the lysis buffer and centrifuged at 15,000 × g for 10 min, and the supernatant mixed with Laemmli buffer and frozen at 80° C.

Electrophoresis was done on the tissue and serum samples under reducing conditions, using 8.75% polyacrylamide gels. All lanes were loaded with 15 µg of protein, and electrophoresis was performed over a period of 1.5 h with a Hoefer minigel system (Hoefer Scientific Instruments, San Francisco, CA). The proteins were electrotransferred with a Transphor Zank transfer unit (Hoefer Scientific Instruments) to a polyvinylidene fluoride membrane (Immobilon-P; Millipore, Bedford, MA) and blocked overnight at 4° C in Tris-buffered saline and 1% polyoxyethylene sorbitan monolaureate (TBST) containing 6% nonfat dry milk. The membranes were washed six times over a period of 30 min in TBST at room temperature. Western blots were made with CL-3, a monoclonal murine antihuman E-selectin antibody (a gift from Dr. C. Wayne Smith, Baylor College of Medicine), LAM 1-14, a murine antihuman L-selectin antibody (a gift from Dr. Thomas Tedder, Duke University Medical Center), and EL-246. Incubation with the primary antibodies was done for 1 h at room temperature in 5% milk at dilutions of 1:1,000 (E-selectin) and 1:500 (L-selectin and EL-246). After multiple washes in TBST, the membranes were incubated with horseradish-peroxidase-labeled goat antimouse IgG antibody (Jackson Laboratories, Bar Harbor, ME) at concentrations of 1:15,000 in TBST with 5% milk. The membranes were then washed six times in TBST, and signal detection was done through electrochemiluminescence (ECL Kit; Amersham Life Sciences, Arlington Heights, IL).

Electron Microscopy and Morphometry

Fixed lungs were cut transversely into 1-cm slices. To obtain a stratified random sample, four 1-cm cubes from the upper and middle lobes, and four 1-cm cubes from the lower lobe were randomly selected. Cubes containing large bronchovascular structures were discarded. Three of these cubes (one chosen randomly from each lobe) were cut into smaller cubes measuring 1 to 2 mm to a side. Ten to 20 of these small cubes were selected at random from each large cube and processed together. The lung cubes were washed in sodium cacodylate buffer and postfixed with 2% osmium tetroxide. The lung cubes were stained en bloc with uranyl acetate, dehydrated in graded ethanol solutions, immersed in propylene oxide, and embedded in Epon resin. The blocks were cut with a diamond knife into thin sections (five per block) and placed on coated, 200-mesh Cu/Rh grids and stained with uranyl acetate and lead citrate. The sections were viewed and photographed on a Philips CM10 transmission electron microscope. Photomicrographs were enlarged to ×8,500 on 11-by-14-inch photographic paper for analysis by a morphometrist blinded to the study conditions.

Thirty micrographs from each of the three lung sites were taken for each animal, for a total of ninety micrographs per baboon. Morphometric analysis was done as previously reported from our institution (24), according to methods described by Weibel and Bolender (25) and Underwood (26). Each photo was point/intercept counted using a 112-line overlay, yielding 224 points per micrograph. Morphometric data were normalized relative to the surface density of epithelial basement membrane in a standard volume for alveolar tissue, as reported previously (24). Morphometric measurements were made for epithelial-cell volume density including alveolar Type I and Type II cells, endothelial-cell volume density, inflammatory-cell volume density in the intravascular, interstitial, and alveolar compartments, and interstitial volume density. The interstitium was analyzed with both matrix and cell counts. Fibroblast volume density data included other noninflammatory interstitial cells that cannot be distinguished from fibroblasts in electron micrographs because of the fragmentary cytoplasmic profiles present. These data are expressed as the ratio of the volume (µm³) to surface area of alveolar basement membrane (µm²). For calculation of total alveolar-basement-membrane surface area, segments of alveolar basement membrane that were denuded of epithelial covering were counted as bare basement membrane and added to the total. In analysis of capillary basement membrane, areas of endothelial fragmentation or displacement were counted as disrupted capillary basement membrane and expressed as percent coverage of the respective alveolar basement membrane.

Data Analysis

Data from the two groups of animals are shown as mean ± SE. Repeated-measures data from physiologic measurements were analyzed through linear regression analysis, allowing for both fixed and random effects. Missing values resulting from animals dying at different times were taken into account with the standard repeated-measures model (SAS statistical software; SAS Institute, Cary, NC). Drug effects and changes in parameters over time were evaluated at the 48-h time point. The results of biochemical assays on BALF and tissue specimens, and the morphometry data, were compared with unpaired t tests. Survival curves were generated using the LIFETEST Procedure on the SAS system. Values of p are provided in the text and tables. A value of p < 0.05 was considered significant, and trends were noted for p < 0.10.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The time-dependent changes in physiologic and hematologic parameters, pulmonary gas exchange, biochemical markers of tissue injury, and animal survival are provided in the following sections. Overall, EL-246 provided no significant protection against the physiologic manifestations of E. coli sepsis or sepsis-induced organ injury. The group of animals treated with EL-246 also had a significantly shorter survival time than the control septic animals (p < 0.05). These data are shown in Figure 1. Median survival for EL-246-treated animals was 24 h after antibody treatment, compared with > 36 h for septic animals that did not receive treatment with the antibody.

View larger version (12K): [in this window] [in a new window]   Figure 1.   Survival curves of control and EL-246 treatment groups. Mortality in the group treated with EL-246 antibody to E- and L-selectin was increased compared with that in the sepsis control group. (A) Sepsis control baboons. (B) EL-246-treated baboons, (*p = 0.019). Arrow represents infusion of live E. coli at t = 12 h.

Physiologic Measurements

The results of the physiologic measurements for the different time points in the experiments are shown in Table 1. Hemodynamic measurements of HR, MAP, CO, DO₂, and SVR revealed a hyperdynamic state typical of septic shock in the untreated control group. The baboons treated with EL-246 had a very similar hemodynamic profile. The CO/kg increased after treatment with HK E. coli, and further increased after treatment with live bacteria (p = 0.001). This increased CO was maintained throughout the studies in the sepsis control group and in the EL-246 group. There was no difference in the two groups' CO response. In both groups there was a decrease in SVR after administration of HK bacteria, with a further continued decrease after infusion of live bacteria that was significant over time, but not statistically different in the two groups. The time-dependent responses in MAP, DO₂, and urine output (UOP) are shown in Figure 2. Both groups had a small decrease in MAP and small increase in HR after infusion of HK bacteria, which progressed over time after infusion of live E. coli. As with the CO, the DO₂ increased after infusion of live bacteria and then slowly returned toward baseline over the remainder of the experimental period, with no difference between groups. UOP was maintained after infusion of HK E. coli in both groups, and decreased after administration of live bacteria. The UOP in the EL-246-treated animals, however, was significantly lower than in sepsis control animals (sepsis control: 3.8 ml/kg/h at 36 h; EL-246: 0.7 ml/kg/h; p = 0.003) (Figure 2). This difference in UOP occurred despite administration of similar volumes of intravenous fluid (data not shown) and maintenance of similar MAP and PCWP in both groups (Table ).

View larger version (15K): [in this window] [in a new window]   Figure 2.   Oxygen delivery (DO2), mean arterial pressure (MAP), and urine output (UOP) in control and EL-246-treated baboons. There was no decrease in DO2 over time or between groups. The MAP decreased significantly over time (p = 0.01), with no difference between groups. Both groups had a significant decrease in UOP over time (p = 0.01), but the UOP was significantly lower in EL-246-treated baboons than in controls (p = 0.0026).

Hematologic values are shown in Figure 3. Hemoglobin values fell progressively during the experiment, with no significant difference between the two groups. There was a precipitous initial decline in WBC count after infusion of live E. coli, which was followed by recovery to baseline values. There was no significant difference between the two groups in this effect. There was progressive thrombocytopenia during sepsis in both groups, with no difference between the groups.

View larger version (14K): [in this window] [in a new window]   Figure 3.   Complete blood counts of EL-246-treated and control baboons during sepsis. There was no difference between the two groups in hemoglobin (Hgb), leukocyte count (WBC), or platelet count during the experiments.

Pulmonary gas exchange and acid-base balance were evaluated with serial arterial blood gas and A/ measurements. At the end point of the study, the EL-246-treated animals had a significantly lower serum pH (p = 0.001) and calculated HCO₃ (p = 0.001) than did the septic controls, with no difference in Pa_CO2, reflecting a more severe metabolic acidosis in the antibody-treated animals. These data are shown in Figure 4 and Table . Both groups had a significant decrease in Pa_O2 over time, but there was no difference between the two groups. A/ relationships are shown in Figure 5. Intrapulmonary shunt expressed as %CO significantly increased as a function of time in both septic groups (p = 0.001). There was a minimally greater increase in shunt in the sepsis control group than in the EL-246 groups (10.6% versus 8.1%), but this effect was not significant. Dead space (VD/VT) increased slightly over time in both groups, and there was a trend toward a greater increase in VD/VT in the EL-246 treated group over time as compared with the sepsis control group (0.44 versus 0.36, p = 0.07).

View larger version (14K): [in this window] [in a new window]   Figure 4.   Changes in arterial pH and [HCO3] in control and EL-246-treated baboons during sepsis. EL-246-treated baboons had a greater decrease in arterial pH than did control animals which was statistically significant (p = 0.01), and a significantly lower serum [HCO3] (p < 0.05).

View larger version (47K): [in this window] [in a new window]   Figure 5.   Ventilation-perfusion (A/) data during sepsis. There was a significant increase over time for sepsis control and EL-246-treated baboons for shunt as percent cardiac output (%CO), and dispersion of perfusion (SDq), with no difference between the two groups. The dead space as a percent of tidal volume (%V T) increased slightly over time in both groups, with a trend toward greater increase in the EL-246-treated group (p = 0.07). Dispersion of ventilation (SDv) was not different between groups.

The two control baboons treated with EL-246 antibody alone maintained stable blood pressure, CO, UOP, pulmonary gas exchange, and acid-base balance. The EL-246 antibody produced a transient decrease in blood neutrophil count in these two animals, but no other evidence of toxicity. These baboons were awakened 24 h after the antibody was administered, and returned to the vivarium in good condition.

EL-246 levels were measured in four baboons after the infusions, to characterize the serum half-life of the drug (Figure 6). The antibody was measurable 1 h after infusion, and declined slowly after a period of 24 h.

View larger version (17K): [in this window] [in a new window]   Figure 6.   Quantitation of circulating antibody levels after infusion of EL-246. After the infusions, EL-246 levels in serum reached or exceeded 10 µg/ml for the duration of the experiments (n = 4).

Biochemical Measurements

The lung wet:dry weight ratios (W/D) were increased in both groups of animals relative to the value in our laboratory for normal baboons of 4.97 ± 0.20 (27). The difference between the slightly higher W/D value of 6.20 in the EL-246-treated group, and the value of 5.75 in the sepsis control group, was not statistically significant (p = 0.58). The small-bowel wet:dry ratios also were increased for sepsis control and for EL-246 treated animals (approximately 6.9 for both groups) with no difference between the two groups. The LDH measurements in BALF were 54.9 IU/L (± 33.7 IU/L) and 28.6 IU/L (± 6.7 IU/L) in EL-246 treated baboons (p = 0.46). The BAL protein was 1.04 mg/ml (± 0.5 mg/ml) and 0.565 mg/ml (± 0.30 mg/ml) in the EL-246 treated animals (p = 0.43).

The results of MPO analysis of various organs, which was used as a marker of PMN content, are summarized in Figure 7A. MPO values were substantially higher in the lung than in other tissues, but MPO values in the lung, kidney, small bowel, and heart were not different between the two groups. There was a higher MPO value for the liver in EL-246-treated animals, which approached significance (p = 0.057). Figure 7B shows the results of the measurement of TBARS used to estimate nonspecific lipid peroxidation as a marker of tissue injury. There was no difference in TBARS between the two groups for kidney, liver, heart, or small bowel, but the TBARS value was significantly lower for the lungs of the EL-246-treated animals (p = 0.036).

View larger version (21K): [in this window] [in a new window]   Figure 7.   Myeloperoxidase (MPO) and thiobarbituric acid-reactive substances (TBARS) measurements on tissues from sepsis control and EL-246-treated baboons. There was no significant difference between the sepsis control and EL-246-treated groups in heart, kidney, small bowel, or lung tissue concentrations of MPO or TBARS. The MPO values were higher in the livers of EL-246-treated baboons (*p = 0.057). TBARS assay shows a significantly lower value in the lungs of EL-246 treated baboons (*p = 0.036). There was no significant difference between sepsis control and EL-246-treated animals for TBARS values of heart, liver, kidney, or small bowel tissue.

Western Blots

Western blot analysis was done on timed serum samples for E- and L-selectin to assess for the appearance of soluble forms of the adhesion molecules in sepsis. These results for one sepsis control animal are shown in Figure 8A and B. E-selectin, seen as a band at 110 kD, was detected in small amounts in the serum of the baboons before the initiation of sepsis. After the infusion of HK E. coli, the serum E-selectin content increased and remained elevated throughout the experimental period (Figure 8A). L-selectin also was detected throughout the experimental period in the serum (Figure 8B), as a dual band at 80 and 100 kDa. Western blot analysis was also done with EL-246 on serum from a septic control baboon to test the ability of this antibody to bind soluble E- and L-selectin. An intense signal at approximately 80 kDa and 100 kDa was present in serum taken at all time points throughout the experiment, a finding most consistent with binding of EL-246 to L-selectin in the serum (Figure 8C). Western blots of lung tissue with anti-E-selectin antibody showed a strong band at 110 kDa in the septic baboons. No band was visible in the lungs of nonseptic ventilated baboons. Western blot analysis with EL-246 antibody showed a strong band at the same molecular weight as the E-selectin band, and a less intense band at 70 kDa, which was consistent with binding of selectin (data not shown).

View larger version (72K): [in this window] [in a new window]   Figure 8.   Western blot analysis of serum from a sepsis control animal at 0 h, 12 h, 13 h, 18 h, 24 h, 36 h, and 48 h with anti E-selectin (A), anti L-selectin (B), and EL-246 antibodies (C ). A soluble form of E-selectin was present as a band at 110 kDa in small amounts at time 0 h, and increased after infusion of heat-killed (HK) and live E. coli at all time points sampled (A). L-selectin is present as a dual band at approximately 80 kDa and 100 kDa at all time points (B). Western blot of serum samples with EL-246 shows strong bands at 80 kDa and 100 kDa, indicating that the antibody reacts with the soluble selectins in the serum at all time points (C ).

Pathology

At the time of autopsy, all 12 animals used in the study showed gross evidence of injury to the lungs, kidneys, liver, heart, and intestine. Focal areas of hemorrhage were seen, but no evidence of metastatic abscess formation was found. Quantitative cultures of tissue specimens, however, were not made. Light microscopy was performed on lung specimens from each group at the time of autopsy. Typical photomicrographs are shown in Figure 9A and B. Low-power views (×400) of lungs from sepsis control (Figure 9A) and EL-246-treated (not shown) baboons showed thickening of the intraalveolar septae, with inflammatory cell infiltration. High-power views (×1,000) showed that in the lung of both septic control (Figure 9B) and EL-246-treated animals (not shown), PMN were adherent to the endothelial surface of small veins and venules. Light microscopy was also performed on kidney samples from each group. Diffuse tubular injury was evident in both groups, and there was deposition of amorphous material in the renal tubules of the EL-246-treated animals that was not present in septic controls (data not shown).

View larger version (108K): [in this window] [in a new window]   View larger version (95K): [in this window] [in a new window]   Figure 9.   Light microscopy of lung specimens from septic control baboons demonstrates inflammatory cells in the intraalveolar septa. Low-power view (A, original magnification: ×400). At ×1,000 (B), PMN are adherent to the endothelium of small venules in the lungs.

Quantitative electron microscopy data from the baboons' lungs are summarized in Table 2. The data are expressed as volume of tissue (µm³) normalized to surface area of alveolar basement membrane (µm²). Both the sepsis control and EL-246 groups had morphologic evidence of significant lung injury. By comparison with normal values for baboons (28), there were increases in normalized volume densities of Type I and II epithelial cells, interstitium, total PMN, and fibroblasts. There was no significant difference between the experimental groups for these indices. There was a significant difference in endothelial-cell volume between the two groups. The endothelial-cell volume was significantly lower in the EL-246-treated group (0.294 µm³/µm²) than in the sepsis control group (0.372 µm³/µm²) (p = 0.036), and was similar to the previously reported value of 0.309 µm³/µm² for normal baboons (28). This finding suggests less endothelial cell edema or injury in the EL-246-treated group. The amount of disrupted capillary membrane for sepsis control and for EL-246-treated animals was similar to that for normal baboons (24). Compartmental analysis of lung PMN indicated no significant differences in the distribution of alveolar or capillary PMN. There was a decrease in interstitial PMN in EL-246-treated animals (p = 0.05), which is shown in Figure 10. The surface area of alveolar epithelial Type I and Type II cells as a percentage of total alveolar surface area was 94.4% and 4.34% for sepsis control and 95.4% and 3.91% for EL-246-treated animals, respectively (Table ). These values did not differ from our values for normal animals of 95.1% and 3.3% (28). The percent bare basement membrane was 1.25% for sepsis control and 0.67% for EL-246-treated animals, which were values similar to previously reported normal values (28).

View larger version (18K): [in this window] [in a new window]   Figure 10.   Distribution of PMN in lung compartments of sepsis control and EL-246-treated baboons, based on data from electron micrographs. There were no significant differences in the PMN content of alveolar or capillary compartments, but decreased interstitial PMN were present in the EL-246-treated group (p = 0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The experiments described in this report were performed to test the hypothesis that pharmacologic blockade of the selectins involved in early leukocyte-endothelial interactions would protect septic primates from lung injury, end-organ injury, and mortality. This hypothesis is based on substantial evidence for involvement of PMN in the pathogenesis of ARDS, and the pivotal role of selectins in recruiting of PMN into organs during inflammation. The use of an antibody with the ability to bind both the L- and E-selectins was expected to provide more complete blockade of PMN influx into organs. Our data show, however, that EL-246, which blocks leukocyte adhesion in vitro, does not completely prevent PMN influx into the lungs of septic baboons, and does not protect against injury to the lung or other organs. Additionally, there was increased metabolic acidosis and decreased survival time in septic baboons treated with EL-246. A unique aspect of this study was pretreatment of these animals with HK E. coli to activate the endothelium, as demonstrated by the increased concentration of soluble E-selectin detectable at 12 h after infusion. The animals then developed acute lung injury and multiple organ dysfunction, and survived for several days after the infusion of live bacteria. This type of sepsis model is more relevant clinically than acute infusions of live bacteria into healthy animals, which result in rapid cardiovascular collapse and early death.

Leukocytes are part of the normal pulmonary defenses, but a derangement of the inflammatory response is likely to be a part of the pathogenesis of ARDS. PMN are capable of releasing products for bacterial killing that may also injure host cells. There is histologic evidence of neutrophil influx in ARDS in humans (4), and BALF of patients with ARDS contains increased numbers of PMN that correlate with gas- exchange abnormalities (3). It is now recognized that selectin-mediated binding contributes to leukocyte recruitment into the alveolar spaces (2, 29). E-selectin is expressed on the endothelial-cell surface after stimulation by LPS or cytokines such as interleukin-1 (IL-1) and tumor necrosis factor- (TNF-), and its ligands are present on leukocytes including PMN, lymphocytes, and eosinophils (5). L-selectin is present constitutively on the surface of leukocytes including lymphocytes, PMN, and monocytes (12). Binding to endothelium by neutrophil L-selectin is stimulated by activation (e.g., with phorbol myristate acetate [PMA] and leukotrienes) (12). L-selectin is shed from the neutrophil with activation and binding to endothelium, leading to increased levels of soluble L-selectin (30).

In most organs, the postcapillary venule is the prominent site of neutrophil rolling and adhesion (6, 31). An in vivo study of E-selectin expression in monkeys infused with LPS showed staining in postcapillary venules, as well as in smaller and larger arteries after 6 h. The pathologic findings in the pulmonary circulation, however, were not detailed (11). After infusion of live E. coli into baboons, E-selectin is expressed within 6 hours in the lung, liver, and kidneys in arteries, arterioles, capillaries, and venules. In contrast, little E-selectin expression was found in baboons with hypovolemic shock (9). The precise distribution of E-selectin in the human pulmonary vasculature is unknown, and its importance in leukocyte trafficking in the lung is uncertain. Because lung capillaries have a diameter similar to that of the PMN, prolonged physical contact of PMN with pulmonary capillary endothelial cells could render loose adhesion-molecule function unnecessary. A requirement for L-selectin, however, has been demonstrated in L-selectin-deficient mice, which have decreased accumulation of neutrophils in noncapillary microvessels with pulmonary inflammation (31). In acute lung injury in rats, induced by IgG immune-complex deposition or complement activation by cobra venom, treatment with antibodies to L-selectin results in decreased lung MPO content and tissue injury (15). A functional role for E-selectin in recruiting PMN in the lung in vivo has also been demonstrated in experimental acute lung injury in rats caused by hindlimb ischemia-reperfusion and immune-complex injury. Treatment of the rats with antibody to E-selectin prior to the insult results in reduced pulmonary neutrophil accumulation and decreased vascular permeability and hemorrhage than in untreated animals (8, 14). Similar results have been demonstrated with blocking antibodies to L-selectin in vivo. In addition, combined blockade of E- and L-selectin in rats with immune-complex-mediated lung injury is more effective than blocking either selectin alone (16).

The effectiveness of the EL-246 antibody in blocking neutrophil adhesion has been demonstrated ex vivo in vascular preparations (17), and the antibody was found to improve arterial oxygen tension and to decrease lung MPO and BAL protein after 6 h in septic pigs (18), as well as to increase survival in sheep with ischemia-reperfusion lung injury (19). For most organs studied in our animals, including the lungs, MPO values were similar in both groups. This finding does not rule out functional blockade of adhesion by the antibody, because the MPO measurement assesses total PMN, including those cells sequestered in capillaries. Although light microscopy of the lung showed adherent neutrophils on the endothelial surfaces of small venules in both the control and EL-246-treated animals, the morphometric data showed decreased interstitial PMN in the EL-246-treated animals, suggesting that the antibody was functional in these studies. Of note are the significantly higher MPO values in the liver in the EL-246-treated group. The explanation for this finding is unknown, but it implies that sequestration of PMN in the liver occurs through non-selectin-based mechanisms.

There are several potential explanations for the incomplete inhibition of neutrophil influx and failure of the EL-246 antibody to protect the lung and other organs. We demonstrated that a single dose of EL-246 produced measurable levels in serum of at least 10 µg/ml, which inhibits adhesion in vitro (17). This makes suboptimal dosing in our study unlikely. The reactivity of EL-246 with baboon serum shown by Western blot analysis suggests that soluble adhesion molecules could compete for the antibody and decrease antibody binding to the membrane-bound selectins intended for targeting. Another potential reason for incomplete blockade is that functional redundancy of adhesion pathways may allow binding of leukocytes despite selectin blockade (16). In addition, the specific adhesion molecules integral to neutrophil recruitment appear to vary among different types of lung injury (16), and the requirement for E- and L-selectin for neutrophil adhesion in acute lung injury from gram-negative sepsis has not been studied previously in detail.

By most physiologic parameters and biochemical measurements, the manifestations of sepsis and organ injury were not modified in the antibody treated group of baboons in our study. At each time point measurements of A/ were not improved in the EL-246-treated group. The increase in V D/VT in EL-246-treated animals could be explained by an effect of the antibody on the number or distribution of intracapillary leukocytes. The morphologic data suggest less endothelial injury in the EL-246-treated group, but this did not translate into any functional improvement. The minor differences in BAL protein, LDH, and lung TBARS in the EL-246-treated group may reflect the shorter survival time of this group. The kidneys showed extensive gross and histologic damage in both groups, and may have had more injury in the EL-246-treated group, since UOP was lower in these animals. The severe metabolic derangement in the baboons treated with the antibody was also an unexpected finding. The profound metabolic acidosis that occurred in the EL-246-treated group was not attributable to differences between groups in blood pressure, CO, or O₂ delivery. The decreased UOP and perhaps more severe renal injury after treatment with EL-246 may explain a portion of this acidosis. The marked pH decline, however, also raises the possibility of a metabolic injury at the cellular level.

To examine the possibility that the antibody per se resulted in toxicity, we treated two control baboons with only the EL-246 antibody at the therapeutic dose. The antibody alone produced a transient decrease in blood neutrophil count in these two animals, but no other evidence of toxicity. These data imply that the deleterious effect of the antibody in the baboon in severe septic shock is related to infusion of the antibody in conjunction with the activated inflammatory state. One possible reason for this effect is that endothelial E-selectin, which was upregulated by the prior dose of HK E. coli, was bound by the infused EL-246. The subsequent infusion of live E. coli could then activate complement, resulting in its fixation by the endothelial-bound antibodies and leading to vascular injury. This may explain differences in our results from the more acute earlier models in which protection by the antibody was demonstrated (18, 19).

Other potential explanations for deleterious effects of EL-246 therapy in sepsis can be inferred from our current understanding of the biology of adhesion molecules. Soluble E-, P-, and L-selectin are detectable in the blood of normal volunteers, and have been found in the plasma of patients with ARDS (30, 32). The presence of E- and L-selectin in the serum of the septic baboons in our study, and the reactivity of EL-246 with serum in the Western blot analysis, suggest that immune-complex formation could be a mechanism of toxicity in these experiments. This possibility is supported by the microscopic appearance of the kidneys, showing diffuse deposition of amorphous material in the tubules, in accord with immune-complex deposition. Preliminary immunohistochemical staining of the kidneys for EL-246 also showed the antibody to be present in some of the tubules. Potentially, this problem could be averted by using an Fab fragment of the antibody. It is also possible that the antibody binds to soluble adhesion molecules and interferes with undefined adaptive functions of these molecules in sepsis. Some data support the concept that soluble adhesion molecules may abrogate inflammation. Soluble L-selectin from human plasma inhibits leukocyte adherence to endothelium (30), and in rats treated with intratracheal LPS, intratracheal administration of soluble E-selectin decreases PMN migration into the alveolar spaces (33).

Detrimental effects of therapy with antiselectin antibody could also result from its binding to adhesion-molecule targets on cells other than PMN, thereby interfering with trafficking or function of lymphocytes and mononuclear cells in sepsis, in which host-defense mechanisms involving these leukocytes are critical. Another consideration is that antibody binding to the selectins could affect endothelial or leukocyte intracellular signaling mechanisms. E-selectin and intercellular adhesion molecule-1 (ICAM-1) may have intracellular signaling functions, since cross-linking of these molecules by antibodies can activate tissue-factor production by the endothelial cell (34). EL-246 could thus facilitate endothelial activation by binding E-selectin, or could activate mononuclear cells by cross-linking of L-selectin, resulting in greater production of tissue factor, nitric oxide, TNF, or other mediators of sepsis, with worsening of the sepsis syndrome.

In conclusion, administration of EL-246, a functional antibody directed against E- and L-selectin, conferred no protection against hemodynamic derangement or lung injury from E. coli sepsis in baboons. Furthermore, UOP was decreased and metabolic acidosis was worsened in EL-246-treated animals, and this group had a significantly shorter median survival time than did untreated septic animals. These experiments show that immunotherapy of septic shock in primates with an anti-E- and L-selectin antibody is not beneficial, and may potentiate injury. These data demonstrate a potential risk of the therapeutic clinical use of monoclonal antibodies to selectins in sepsis. Most septic patients would be expected to have upregulated the expression of these adhesion molecules at the time of intervention, possibly putting them at risk for adverse consequences of the therapy.