Neutrophil Elastase Inhibitor, ONO-5046, Modulates Acid-induced Lung and Systemic Injury in Rabbits

Specific pathogen-free Japanese White rabbits (range of weight, 3-4 kg) were anesthetized with pentobarbital (25 mg/kg), and anesthesia was maintained with 0.5% halothane. A 20-gauge intravenous catheter was inserted into an ear vein for administering fluid and drugs. A catheter was inserted into the carotid artery to monitor blood pressure and for blood sampling. An endotracheal tube was inserted through the tracheotomy. The rabbits were in the supine position, and their lungs were ventilated with a constant-volume pump (Harvard, Millis, MA) with an inspired oxygen fraction of 1.0 and a positive end-expiratory pressure of 3 cmH²O. The tidal volume was adjusted to maintain an arterial P^CO2between 35 and 45 mmHg. Two ml [center dot] kg sup -1 [center dot] h sup -1 of lactated Ringer's solution with pancuronium (2 mg/h) was administered intravenously throughout the experiment.

These animal experiments were done in conformity with the "Guiding Principles for Research Involving Animals" of the Yokohama City University School of Medicine, and the protocols were approved by the animal care committee.

All the rabbits were placed in the right decubitus position to facilitate liquid deposition into the right lung.

Control Group (n = 12). An intravenous infusion of normal saline (1 ml/kg) was started; 15 min after this event, 1.2 ml/kg of phosphate buffered saline (PBS; pH, 7.4) mixed with Evan's blue dye was delivered into the right lung (primarily the right lower lobe) using a 10-ml syringe and a pediatric feeding tube. The intravenous infusion of saline was continued throughout the experiment.

HCl Group (n = 12). Fifteen minutes after the initiation of an infusion of normal saline (1 ml/kg), the acid instillate, HCl (pH, 1.25; volume, 1.2 ml/kg) mixed with Evan's blue dye, was delivered into the right lung. HCl (0.1 N) was diluted to a pH of 1.25 with normal saline. This quantity of acid was instilled because it had been shown to cause significant lung damage but did not lead to the death of the animals. [6,11]The placement of the instillate in the lung was confirmed by the visualization of the Evan's blue dye at post-mortem.

ONO Group (n = 12). ONO-5046 (kindly provided by ONO Pharmaceutical, Osaka, Japan) was dissolved in sterile normal saline to a concentration of 10 mg/ml and filtered through a 0.45-mm filter (Millipore, MA) before injection. Fifteen minutes before HCl instillation, ONO-5046 (10 mg/kg) was administered intravenously and then continuously infused (10 mg [center dot] kg sup -1 [center dot] h sup -1, dissolved in the saline) throughout the experiment. This dose of ONO-5046 was chosen based on an investigation done in endotoxemic rabbits. [12]Each animal received the same volume of instillate and the same quantity of intravenous fluid.

Blood samples to measure arterial blood gases and for leukocyte counts were obtained before the infusion of normal saline for the control and HCl groups or before the infusion of ONO-5046 for the ONO group, before the acid instillation, and 30 min, 1, 2, 4, and 8 h after the instillation of fluid into the right lungs. The animals remained anesthetized, ventilated, and monitored for the experimental interval. Eight hours after the instillation of acid or PBS, the rabbits were deeply anesthetized, their abdomens and chests were opened, and the rabbits were exsanguinated.

Six animals in each group were used solely for the determination of the W/Ds. In the other six animals in each group, a 1-g sample of the kidney, liver, small intestine, and heart were harvested for the measurements of myeloperoxidase (MPO) activity. One-half of the lower lobes of the right and left lungs were clamped and removed for MPO measurement. The remainder of the lungs were used for bronchoalveolar lavage (BAL). BAL of the left and then the right lungs was performed separately with 5 ml of normal saline per lung (three times) using a 3.5-mm (inner diameter) tracheostomy tube. Aliquots of the BAL fluid (BALF) were used for leukocyte counts, and the remainder of the BALF was centrifuged at 1,500g for 20 min at 4 [degree sign] Celsius, and these supernatants were frozen at -70 [degree sign] Celsius. These supernatants were subsequently used for measurements of protein concentration, using the Lowry method, [13]and for elastase assay (to be described). Leukocytes in the BALF and blood were quantified using Gentian Violet and Wright-Giemsa stains and a hemocytometer.

Activity of this neutrophil enzyme was used to quantitate polymorphonuclear neutrophil (PMN) sequestration in tissue; this assay has been found to be a more sensitive method for the detection of entrapped neutrophils than quantitative histology. [14]One gram of tissue blotted dry was homogenized in 10 ml of 0.01 M potassium phosphate buffer (PPB; pH, 7.4) containing 1.0 mM ethylene diamine tetraacetic acid (EDTA). Two milliliters of homogenate and 5 ml of 0.01 M PPB containing 1.0 mM EDTA were mixed gently and then centrifuged at 10,000g for 20 min at 4 [degree sign] Celsius. The pellet was rehomogenized in 5 ml of 0.05 M PPB (pH, 6.0) containing 0.5% hexadecyltrimethylammonium bromide. This suspension was freeze-thawed and sonicated with a Branson cell disrupter at 65 W for 1 min. A 0.1-ml aliquot was mixed with 0.79 ml of 0.08 M PPB (pH, 5.4) and 0.1 ml of 16 mM tetramethylbenzidine dissolved in N,N-dimethylformamide at 37 [degree sign] Celsius. After 2 min. 0.01 ml of 30 mM H²O²was added. After incubating 3 min at 37 [degree sign] Celsius, 0.05 ml of catalase solution (300 mg/ml) was added. The mixture was diluted with 4 ml of 0.2 M sodium acetate (pH, 3.0) and then centrifuged at 12,000g for 10 min at 4 [degree sign] Celsius. The supernatant was analyzed by a spectrophotometer (UV-2200, Shimadzu, Kyoto, Japan). One unit of MPO activity was defined arbitrarily as the amount of enzyme necessary to catalyze an increase in absorbance of 1.0/min at 655 nm at 37 [degree sign] Celsius.

Freshly harvested parts of the hearts, livers, kidneys, and small intestines were weighed, dried at 60 [degree sign] Celsius for 72 h, and weighed again. The W/Ds were calculated as wet weights/dry weights. The W/Ds of the lungs also were measured using the method of Selinger et al. [15]to calculate the extravascular lung waters.

The quantification of neutrophilic elastase activity in BALF was determined using the method of Barnett et al., [16]with a specific substrate, N-succinyl-(L-alanine)3-p-nitroanilide (Sigma, St. Louis, MO), for elastase. Twenty-five microliter aliquots of BALF were incubated in a 96 well plate at room temperature for 3 days with 1 mM substrate, 0.1 M Hepes, 0.5 M NaCl, pH of 7.5 in a total volume of 150 ml. Absorbance was measured at 405 nm using a microplatereader (Model 450, BIORAD, Hercules, CA). Elastase activity in the sample was determined from a calibration curve obtained using purified human neutrophilic elastase (Wako, Osaka, Japan).

All data are presented as mean +/- SD. The data between the groups were analyzed by ANOVA and Scheffe's test. The within-group data were analyzed by Student's paired t test. We accepted a P < 0.05 as statistically significant.

There were no significant differences in the mean arterial pressures, heart rates, or in the arterial pH, or Pa^CO2values measured in the three experimental groups (Table 1). Eight hours after the instillation of HCl, the arterial Pa^O2had decreased significantly from 547 +/- 30 mmHg to 338 +/- 138 mmHg in the HCl group; in contrast, the arterial Pa^O2did not change in the control group or in the ONO pretreatment group.

The peripheral blood neutrophil counts over the 8-h experimental interval are shown in Figure 1. The neutrophil counts in the animals that received acid instillations transiently decreased from their baseline counts; the decrease occurred 30 min after the instillation of the acid. Pretreatment with ONO-5046 inhibited this decrease in the number of circulating neutrophils.

The transient decrease of neutrophils was followed by a subsequent increase in the number of circulating neutrophils; the circulating neutrophil counts became elevated 2-8 h after the instillation of the PBS in the control group animals; the circulating neutrophil counts were elevated 4-8 h after the acid instillation in the animals in the ONO group, and the circulating neutrophils were significantly elevated only by the eighth hour after the instillation of acid in the animals in the HCl group. The increase in the circulating neutrophil counts in the control group may have been a result of the instillation of PBS or of the 8 h of anesthesia.

The instillation of acid in the HCl and ONO groups was associated with an increase in the MPO activities in both of the lungs and in the small intestines (Figure 2). The MPO activities in the hearts, kidneys, and livers in these animals were not different from the MPO activities found in these organs in the control animals. The ONO-5046 pretreatment did not affect the myeloperoxidase activities measured in any of the organs.

The instillation of acid increased the W/D ratios of both lungs in the HCl group (Figure 3). Pretreatment with ONO-5046 prevented the increase in the W/D ratios in the noninstilled lungs of the ONO group. The W/D ratios of the hearts, livers, and kidneys in the three experimental groups did not differ; the W/D ratios of the small intestines in the HCl group were significantly greater than those measured in the control and ONO groups.

The protein concentrations of the BALF obtained from the acid-instilled lungs from the animals of the HCl and the ONO groups were significantly increased compared with the protein concentration of the BALF obtained from the PBS-instilled lungs from the control animals (Figure 4). The protein concentrations of the BALF obtained from the noninstilled lungs from the three groups of experimental animals were not different. The ONO-5046 pretreatment did not significantly decrease the protein concentration of the BALF obtained from the HCl-instilled lungs.

The neutrophil counts in the BALF are shown in the Table 2. The BALF from the acid-instilled lungs had neutrophil counts of 85 +/- 15 x 10⁶/ml compared with the counts from the BALF of the control group (5 +/- 0.6 x 10⁶/ml). ONO-5046 pretreatment did not decrease the neutrophil counts in the BALF (69 +/- 23 x 10⁶/ml). In the BALF from the noninstilled lungs, there were 26 +/- 8 x 10⁶/ml neutrophils in the BALF from the animals in the HCl group, and the neutrophil counts were similar in the BALF from the animals in the ONO group (19 +/- 12 x 10⁶/ml).

There was no elastase activity in the BALF from the lungs of the animals in the control group. The elastase activity in the BALF from the acid-instilled lungs in the HCl animals was 3.5 +/- 0.2 U/ml and 1.8 +/- 0.3 U/ml in the BALF from the contralateral lung. There was no elastase activity detected in the BALF from the acid-instilled or from the noninstilled lungs from the animals in the ONO-5046 group.

In this study, we showed that a specific neutrophil elastase inhibitor, ONO-5046, attenuated some of the pathophysiologic effects of acid-induced lung and small intestine injury. The ONO-5046 pretreatment prevented the increases in the W/Ds of the noninstilled lungs and of the small intestines and normalized the Pa^O2in rabbits who had been exposed to the unilateral acid instillations. Lung histologic sections confirmed that the lung edema was reduced in the ONO-5046 pretreated animals. Notably, these effects occurred despite the fact that the ONO-5046 pretreatment did not affect the neutrophil sequestration in the lungs or small intestines, nor did the pretreatment affect myeloperoxidase activities in the lungs or the necrosis seen on the histologic sections.

Activation of neutrophil and endothelial adhesion molecules are thought to contribute to the increased endothelial permeability in the noninstilled lungs [3,4]in acid-induced unilateral lung injury experiments. There are CD18-dependent and CD18-independent pathways that activate neutrophil adherence. Doerschuk et al. [17]showed that the anti-CD18 monoclonal antibody, 60.3, did not inhibit neutrophil emigration into acid-instilled lungs, suggesting that CD18-independent pathways might be more important in acid-induced neutrophil sequestration in the lungs. Neutrophil stiffness and ICAM and other selectins have been found to contribute to neutrophil sequestration in acid-injured lungs. [18]However, this investigation suggests that lung endothelial permeability can be normalized despite the fact that neutrophil sequestration still occurs; blockade of neutrophil elastase function was sufficient to normalize the endothelial permeability and the oxygenation defects in acid-exposed animals.

The number of circulating neutrophils decreased 30 min after the instillation of HCl and then increased gradually during the 8-h experiment. These results are consistent with those reported by Goldman et al. [3]Recent reports have suggested that adhesion receptor families (i.e., selectins and beta²integrins) promote early neutrophil sequestration after the onset of sepsis. [19]The neutropenia that occurred 30 min after the instillation of acid in this study may have resulted from L-selectin and E- or P-selectin interactions; pretreatment using ONO-5046 modulated this early neutropenia, suggesting perhaps that the blockade of neutrophil elastase may affect adhesion molecules. This is speculative; there may be other mechanisms for the neutropenia, and this investigation did not specifically address this issue.

The neutral protease, elastase, is released from the azurophilic granules of activated neutrophils. The elastase has broad substrate specificity that includes basal membrane proteins, cell receptors, fibronectin elastin, collagens, and proteoglycans. [20]This elastase also has been found to cause endothelial cell injury [21]and to proteolytically destroy lung surfactant proteins. [22] 

ONO-5046 is a synthetic compound and is a reversible and competitive inhibitor of human, rabbit, rat, hamster, and mouse neutrophil elastase. [9]ONO-5046 has a much lower molecular weight than alpha¹-protease inhibitor (alpha¹-PI) and may exert its effects in the microenvironment between neutrophils and tissues, whereas alpha¹-PI incompletely blocks neutrophil-mediated tissue destruction. Inhibition of neutrophil elastase has been shown to be useful in other models of lung injury. Recently, Yoshimura et al. [23]reported that the administration of ONO-5046 inhibited leukotriene B4-induced lung injury in isolated rabbit lungs. The administration of ONO-5046 was also reported to be effective in modulating endotoxin-induced lung injury in sheep and guinea pigs. [10,12]Sakamaki et al. [12]demonstrated that in a guinea pig model of endotoxin-induced acute lung injury, ONO-5046 did not attenuate the increase in the permeability of the endothelium, but rather it appeared to protect the alveolar epithelium. A possible explanation for the discrepancy between the lack of protection of the epithelium by ONO-5046 in the present investigation is that acid instillation directly injures the epithelium, unlike the instillation of endotoxin.

In summary, we and others have shown that several agents have effects when used as pretreatments in animal models of acid-induced lung injury. The present experiments demonstrate that the administration of ONO-5046, a neutrophil elastase inhibitor, significantly improved oxygenation and improved lung edema induced by unilateral acid instillation. The utilization of this drug or others that decrease the inflammatory response in the clinical setting will depend on whether the drugs cause significant side effects or problems in host defense. Future experiments need to focus on whether this agent is useful when it is administered after the acid injury has occurred.