Journal of Applied Physiology AJP: Lung Cellular and Molecular Physiology
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J Appl Physiol 82: 1743-1750, 1997;
8750-7587/97 $5.00
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Journal of Applied Physiology
Vol. 82, No. 6, pp. 1743-1750, June 1997
PULMONARY CIRCULATION AND LUNG FLUID BALANCE

Inhibition of CD18 or CD11b attenuates acute lung injury after acid instillation in rabbits

Hans G. Folkesson and Michael A. Matthay

Cardiovascular Research Institute, University of California, San Francisco, California 94143-0130

ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES

ABSTRACT

Folkesson, Hans G., and Michael A. Matthay. Inhibition of CD18 or CD11b attenuates acute lung injury after acid instillation in rabbits. J. Appl. Physiol. 82(6): 1743-1750, 1997.---Acid-induced lung injury is mediated primarily by activated neutrophils. Although a prior study demonstrated that acid-induced neutrophil influx into the air spaces was not CD18 dependent, we hypothesized that either a neutralizing anti-CD18 monoclonal antibody (MHM23) or a neutrophil inhibitory factor (NIF), NIF (CD11b,18), might attenuate acid-induced lung injury in rabbits by interfering with neutrophil activation. This hypothesis derived from in vitro studies that reported that anti-CD18 therapy prevented tumor necrosis factor-alpha -induced neutrophil activation. Hydrochloric acid (pH = 1.5 in one-third normal saline) or one-third normal saline (4 ml/kg) was instilled into the lungs of ventilated, anesthetized rabbits. The rabbits were studied for 6 h. In acid-instilled rabbits without the anti-CD18 monoclonal antibody or NIF (CD11b,18), severe lung injury developed. In acid-instilled rabbits, pretreatment (5 min before acid) with the anti-CD18 monoclonal antibody (2 mg/kg iv) or pretreatment with the NIF (anti-CD11b,18, 10 mg/kg iv) prevented 50-70% of acid-induced abnormalities in oxygenation, the increase in extravascular lung water, and extravascular protein accumulation. The anti-CD18 monoclonal antibody was associated with a significant increase in air space neutrophils by bronchoalveolar lavage, suggesting that the neutrophils respond normally to chemotactic stimuli but that the neutrophils did not injure the lung even though they accumulated in the air spaces. In summary, neutralization of CD18 attenuates the acute lung injury after acid instillation without reducing the number of neutrophils in the air spaces, suggesting that anti-CD18 therapy may be beneficial because of its capacity to reduce neutrophil activation.

pulmonary edema; hydrochloric acid; lung endothelial permeability; neutrophil inhibitory factor

INTRODUCTION

ASPIRATION OF GASTRIC CONTENTS is one of the most common clinical events associated with development of the adult respiratory distress syndrome (ARDS); the mortality for ARDS resulting from acid aspiration ranges from 40-50% (10, 33). Although acid itself may directly injure the lung, acid aspiration-induced acute lung injury is primarily mediated by activated neutrophils. In several studies, acid-induced lung injury has been reduced by either blocking neutrophil products, depleting neutrophils, or inhibiting their migration into the air spaces (9, 12, 14, 15, 19, 20, 22, 27, 37). Acid aspiration induces neutrophil recruitment into the lung by the release of macrophage- and epithelial cell-derived chemotactic and inflammatory molecules, especially interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-alpha ) (12, 14, 15, 19, 27). Once recruited to the lung, activated neutrophils may induce injury on binding to or migrating through the pulmonary capillary endothelium (11, 21, 29).

We recently reported that the neutrophil chemoattractant IL-8 mediates the development of acid-induced acute lung injury in rabbits (9). In that study, treatment with a neutralizing monoclonal anti-IL-8 antibody reduced neutrophil influx into the air spaces and reduced the extent of lung injury by 70%. It is also known that anti-TNF-alpha therapy can reduce neutrophil accumulation in the lung and the severity of lung injury after acid aspiration-induced lung injury (15). In other studies, it has been established that IL-8 upregulates neutrophil beta 2-integrins for endothelium (6, 34) and TNF-alpha also upregulates adhesion molecules on endothelium and neutrophils (5, 24, 39). The neutrophil adhesion complex CD11,18 (LFA-1/Mac-1/p150,95) (4) mediates stimulated neutrophil adhesion to the endothelium (17). The CD11,18 family of integrins is necessary for normal leukocyte trafficking in the systemic circulation, although it is not always required for neutrophil adhesion and sequestration in the lung (7). The requirement of CD11,18 for neutrophil adhesion and migration in the lung is dependent on the stimulus and the route by which an inflammation is induced (7). Patients lacking CD11,18 integrins on their circulating monocytes and neutrophils are defective in several functions specific to these cells (16, 40). The involvement of CD18 integrins in neutrophil influx after hydrochloric acid instillation in the lung was studied initially in rabbits by Doerschuk et al. (7). In that study, it was suggested that CD18 may not be involved in mediating acid injury to the lung because anti-CD18 therapy did not inhibit acid-induced influx of neutrophils into the air spaces of the lungs. However, the magnitude of lung injury was not examined in that study.

Interestingly, Nathan and Sanchez (31) reported that anti-CD18 therapy prevented a TNF-alpha -induced decline in adenosine 3',5'-cyclic monophosphate (cAMP) in human neutrophils; the decline in cAMP was necessary for TNF-alpha to induce a respiratory burst in neutrophils. Therefore, the primary hypothesis of this study was to determine whether anti-CD18 would reduce the extent of acute lung injury after acid aspiration even though it would not be expected to reduce influx of neutrophils into the air spaces of the lungs. To answer the question, the first purpose of these studies was to determine whether pretreatment with an anti-CD18 antibody would reduce the magnitude of acute lung injury after acid aspiration. The second purpose was to investigate the effects of a naturally occurring hookworm glycoprotein [neutrophil inhibitory factor (NIF; CD11b,18 inhibitor)] on acid-induced acute lung injury. This glycoprotein has been shown to inhibit neutrophil function by binding to the CD11b,18 integrin (30). The studies were designed to measure the three critical indexes of acute lung injury: gas exchange, the magnitude of pulmonary edema, and lung endothelial barrier permeability to protein.

METHODS

Animals, surgical preparations, and ventilation. Male New Zealand White rabbits (n = 34; wt 2.5-3.5 kg, Nitabell, Hayward, CA) were surgically prepared as described earlier (8, 9). Briefly, the rabbits were initially anesthetized by using 4% halothane in 100% O2; the anesthesia was then maintained with 0.8% halothane in 100% O2. Pancuronium bromide (0.3 mg · h-1 · kg body wt-1; Pavulon, Organon, West Orange, NJ) was given intravenously for neuromuscular blockade.

A 22-gauge Angiocath (Deseret Medical, Becton-Dickinson, Sandy, UT) was inserted in the marginal ear vein for administering fluid and drugs. A PE-90 catheter (Clay Adams, Becton-Dickinson, Parsippany, NJ) was inserted in the right carotid artery to monitor systemic blood pressure and to obtain blood samples. A 4.0-mm-inner diameter endotracheal tube was inserted through a tracheostomy. The rabbits were maintained in the prone position during the experiments, ventilated with a constant-volume piston pump (Harvard Apparatus, Dover, MA) with an inspired O2 fraction of 1.0 and with a peak airway pressure of 15-18 cmH2O during the baseline period, and supplemented with a positive end-expiratory pressure of 4 cmH2O. During the baseline period, the respiratory rate was adjusted to maintain the arterial PCO2 between 35 and 40 Torr. Thereafter, the ventilator settings were kept constant throughout the experiment.

The protocol for the studies was approved by the University of California, San Francisco, Animal Research Committee.

Preparation of instillate. A solution of 100 mosmol/kgH2O of NaCl (one-third normal saline) was prepared with isotonic 0.9% saline and distilled water. The 100 mosmol/kgH2O osmolality was chosen to match the osmolality of gastric aspirates. Then, HCl was added to the solution and titrated to a pH of 1.5. In the negative control studies, one-third normal saline was used as the instillate. Evans blue dye (1 mg; Aldrich Chemical, Milwaukee, WI) was added to all instillates to confirm at postmortem examination that the instilled fluid was distributed equally to both lungs.

Generation of monoclonal antibody to CD18 and glycoprotein NIF (CD11b,18). The monoclonal antibody (MHM23) to CD18 is a mouse immunoglobulin G2a (IgG2a) antibody (kindly provided by Genentech, South San Francisco, CA) that recognizes human and rabbit CD18. The antibody preparation was sterile filtered and endotoxin was undetectable by the Limulus polyphemus assay. This monoclonal antibody has in earlier studies been demonstrated to inhibit human lymphocyte antigen-restricted lysis of virus-infected target cells from cytotoxic T lymphocytes and natural killer cell cytotoxicity (18, 36). It has also been shown to protect the tissue in the rabbit ear model of ischemia-reperfusion (23).

Generation of the glycoprotein NIF (CD11b,18) from canine hookworms has been described in detail previously (30). After generation and isolation, the coding region for NIF was then cloned into a plasmid vector and expressed in Pichia pastoris. Recombinant NIF (kindly provided by Corvas) was then obtained from these clones and was used in these experiments. NIF (CD11b,18) has been shown to inhibit neutrophil adhesion to fibrinogen in vitro, an event that is known to be dependent on CD11b,18 (1).

General experimental protocol. In all experiments, after surgical preparations, a 1-h baseline of stable heart rate, systemic blood pressure, and arterial blood gases was required before instillation. Fifteen minutes into the baseline period, 3 µCi 131I-labeled human serum albumin (Frosst Laboratories) was injected intravenously as a vascular tracer protein. Blood samples were obtained every 15 min for the remaining 45 min of the baseline period. The vascular tracer was used to calculate the flux of plasma protein into the extravascular spaces of the lung.

For the instillation, a tubing (5-Fr, Accumark Premarked Feeding Catheter, Concord/Portex, Keene, NH) was gently passed through the tracheal tube until it was placed ~1 cm above the carina. Then, HCl or one-third normal saline (4 ml/kg body wt) was instilled into both lungs over 3 min. After the instillation was completed, the tubing was withdrawn.

Thirty minutes after the instillation, and hourly thereafter during the 6-h experimental periods, blood was sampled.

At the end of the 6-h experiments, the abdomen was opened and the rabbit was exsanguinated by transection of the abdominal aorta. The lungs were removed through a median sternotomy. An alveolar sample was aspirated via a sampling catheter gently passed through the trachea to a wedged position in a distal airway. Then, the left lung was clamped at the main bronchus for later use in extravascular lung water and tracer protein measurements (see below). The right lung was then lavaged two times by using 6 ml of isosmolar 0.9% NaCl containing 12 mM lidocaine (Sigma Chemical, St. Louis, MO) each time.

The radioactivity of the samples was measured. Total and differential cell counts were measured on the blood and bronchoalveolar lavage samples. The total cell counts were calculated as cells per milliliter lavage multiplied by the lavage volume used (12 ml).

By trichloroacetic acid precipitation of the instillates and selected samples from each experiment, it was established that the vascular tracer 131I remained >98% bound to protein.

Specific experimental protocol. There were six experimental groups. Five of these groups received HCl instillation, and one received one-third normal saline instillation.

In the positive control group (n = 7), 5 min before the HCl instillation, the rabbits received 0.9% NaCl (2 ml/kg body wt). Then, the HCl was instilled and the rabbits were studied for 6 h.

In the control antibody group (n = 3), three rabbits were injected intravenously with a control monoclonal antibody (2 mg/kg body wt) of the same isotype as MHM23 (IgG2a) and directed against the gp120 envelope protein on the human immunodeficiency virus (3) and studied for 6 h.

In the CD18-pretreatment group (n = 5), 5 min before the HCl instillation, the rabbits received the monoclonal antibody against CD18 (MHM23; 2 mg/kg body wt iv) and were studied for 6 h.

In the NIF (CD11b,18)-pretreatment group (n = 4), 5 min before the HCl instillation, the rabbits received the recombinant NIF (10 mg/kg body wt iv) and were studied for 6 h.

In the negative control group (n = 4), 5 min before the one-third normal saline instillation, the rabbits received 0.9% NaCl (2 ml/kg body wt iv) and were studied for 6 h.

In the neutrophil-depleted group (n = 3), all circulating neutrophils had first been depleted by administration of vinblastine sulfate (0.75 mg/kg body wt; Sigma Chemical) in the marginal ear vein 4 days before the experiment. On the day before the experiment, the depletion of circulating neutrophils was confirmed by finding no neutrophils on a blood smear stained with modified May-Grünewald Giemsa. On the day of the experiment, the rabbits received the HCl instillation and were studied for 6 h.

Hemodynamics, airway pressure, and arterial blood gases. The heart rate, systemic blood pressure, and airway pressure were measured by using calibrated pressure transducers (Pd23 ID, Gould, Oxnard, CA) and recorded continuously on a Grass polygraph (model 7; Grass Instruments, Quincy, MA). Arterial blood gases and pH and the systemic arterial pressure were measured every 30 min. The alveolar-arterial O2 tension difference was calculated.

Extravascular lung water. Our method for the determination of extravascular lung water has been described previously in detail (2, 38). In brief, the left lung was homogenized and the extravascular lung water was determined by measuring the extravascular water-to-dry weight ratio (g H2O/g dry lung). Because the right lung was lavaged for cell counts, data for extravascular lung water were obtained for the left lung only. The bronchoalveolar lavage from the right lung and the homogenates from both lungs were used for measurement of radioactivity (see below).

Lung vascular permeability. For measurement of lung endothelial permeability to protein, the clearance of the vascular tracer protein 131I-albumin across the endothelium into the extravascular compartments of the lungs was measured. The total extravascular 131I-albumin accumulation in the lung was calculated by taking the total lung 131I-albumin (in lung homogenate and in the alveolar samples) and subtracting the vascular space 131I-albumin. The 131I-albumin in the vascular space was calculated by multiplying the mean 131I-albumin counts in the plasma by the calculated plasma volume in the lungs, as we have done previously (2, 38). The extravascular accumulation of 131I-albumin in the lung was expressed as plasma equivalents or the milliliters of plasma that would account for the radioactivity in the lung.

Statistical analysis. One-way analysis of variance with repeated-measurements analysis was used to compare samples obtained at several time points from the same animal. One-way analysis of variance (factorial) was used when other single groups were compared. The Student-Newman-Keuls test was used as a post hoc statistical test. Values are expressed as either means ± SD or means ± SE as indicated in tables and figure legends.

RESULTS

Oxygenation, ventilation, and pH. There were no effects on the increase in alveolar-arterial O2 tension difference from the administration of the control antibody compared with the positive control group instilled with HCl alone. The alveolar-arterial O2 tension difference in the CD18-pretreatment group was significantly lower than that in both the positive control group (HCl alone) and the control antibody group by 2 h after the acid instillation and remained lower for the 6-h experiment (Fig. 1, Table 1). Furthermore, in the CD18-pretreatment group, the alveolar-arterial O2 tension difference was not significantly different from that in the negative control group (Fig. 1, Table 1). In the NIF (CD11b,18)-pretreatment group, the alveolar-arterial O2 tension difference was lower than in both the positive control and the control antibody groups but elevated compared with the negative control group and tended to increase over 6 h (Fig. 1, Table 1). In the neutrophil-depleted group, the alveolar-arterial O2 tension difference was also significantly lower than in both the positive control and the control antibody groups but higher than in the CD18-pretreatment and negative control groups (Fig. 1). The alveolar-arterial O2 tension difference in the neutrophil-depleted group was similar to that in the NIF (CD11b,18)-pretreatment group (Fig. 1).
Fig. 1. Alveolar-arterial O2 tension difference in positive control, control antibody, CD18-pretreatment, neutrophil (PMN)-depletion, negative control, and neutrophil inhibitory factor [NIF (CD11b,18)]-pretreatment groups over 6 h. Values are means ± SE. The alveolar-arterial O2 tension difference in CD18-pretreatment group was significantly less than that in positive control and control antibody groups from 2 h onward and was no different from that in negative control group. In NIF (CD11b,18)-pretreatment group, alveolar-arterial O2 tension difference was lower than in positive control group but higher than that in negative control group. The alveolar-arterial O2 tension difference in PMN-depleted rabbits was significantly different from negative control and CD18-pretreatment groups but not different from NIF (CD11b,18)-pretreatment group. * P < 0.05 vs. negative control group. dagger  P < 0.05 vs. CD18-pretreatment group. Dagger  P < 0.05 vs. PMN-depleted group. # P < 0.05 vs. NIF (Cd11b,18)-pretreatment group.
[View Larger Version of this Image (26K GIF file)]

Table  1.   Oxygenation, ventilation, arterial pH, systemic blood pressure, heart rate, and airway pressure in experimental groups
Condition n Alveolar-Arterial O2 Tension Difference, Torr Arterial PCO2, Torr pH Mean Systemic Arterial Pressure, mmHg Heart Rate, beats/min Peak Airway Pressure, cmH2O
Positive control group (HCl) 7
  Baseline 106 ± 30  37 ± 4  7.46 ± 0.05  68 ± 13  300 ± 30  18 ± 3 
  6 h After instillation 377 ± 146* 68 ± 20* 7.22 ± 0.12* 56 ± 21  285 ± 23  24 ± 7*
HCl, pH = 1.5 + pretreatment with nonspecific Mab 3
  Baseline 105 ± 33  37 ± 3  7.40 ± 0.06  60 ± 12  305 ± 37  15 ± 3 
  6 h After instillation 440 ± 117* 65 ± 2* 7.11 ± 0.15* 57 ± 17  287 ± 43  31 ± 13*
Pretreatment group (MAb CD11, CD18, HCl) 5
  Baseline 105 ± 41  35 ± 4  7.42 ± 0.03  58 ± 9  300 ± 35  14 ± 4 
  6 h After instillation 132 ± 48dagger 42 ± 4*, dagger 7.36 ± 0.04dagger 60 ± 9  288 ± 48  18 ± 2*
Pretreatment group (NIF, HCl) 4
  Baseline 109 ± 15  35 ± 6  7.44 ± 0.05  59 ± 6  299 ± 28  15 ± 4 
  6 h After instillation 269 ± 119dagger 50 ± 6*, dagger 7.32 ± 0.02dagger 60 ± 5  280 ± 20  19 ± 3*
Neutrophil-depletion group (HCl) 4
  Baseline 111 ± 54  38 ± 3  7.47 ± 0.07  59 ± 9  301 ± 44  16 ± 2 
  6 h After instillation 280 ± 62dagger 32 ± 6*, dagger 7.36 ± 0.05dagger 62 ± 8  290 ± 33  20 ± 3*
Negative control group (1/3 normal saline) 4
  Baseline 108 ± 31  37 ± 1  7.41 ± 0.09  61 ± 4  300 ± 29  17 ± 2 
  6 h After instillation 109 ± 51dagger 41 ± 3*, dagger 7.38 ± 0.05dagger 62 ± 12  287 ± 21  20 ± 2*, dagger
Values are means ± SD; n = no. of rabbits. MAb, monoclonal antibody; NIF, neutrophil inhibitory factor. * P < 0.05, analysis of variance (ANOVA), repeated measures, with Student-Newman-Keuls post hoc test vs. baseline. dagger P < 0.05, ANOVA with Student-Newman-Keuls post hoc test vs. positive control rabbits.

The acid-induced abnormalities in arterial PO2 and pH were mostly prevented by pretreatment with monoclonal antibodies against CD18 or NIF (CD11b,18) as well as by neutrophil depletion (Table 1).

Extravascular lung water. There were no differences in extravascular lung water between the rabbits instilled with HCl alone and the rabbits given the control antibody. The extravascular lung water (g H2O/g dry lung) in the CD18-pretreatment group was 35% lower (P < 0.05) than in both the positive control and the control antibody groups (Fig. 2). In the NIF (anti-CD11b,18)-pretreatment group, the mean extravascular lung water was 15% lower, a value that did not reach statistical significance (Fig. 2). In the neutrophil-depleted group, the extravascular lung water was also not different from that in the negative control group (Fig. 2).
Fig. 2. Extravascular lung water in positive control, control antibody, CD18 pretreatment, PMN-depletion, negative control, and NIF (CD11b,18)-pretreatment groups at 6 h. Data are means and SD. Extravascular lung water in CD18-pretreatment and NIF (CD11b,18)-pretreatment groups was 35 and 15% (NIF not statistically different) less, respectively, than in positive control and control antibody groups and was no different from that in negative control group. Extravascular lung water of a normal uninstilled rabbit lung is 3.2 g H2O/g dry lung. * P < 0.05 vs. positive control group. dagger  P < 0.05 vs. control antibody group.
[View Larger Version of this Image (19K GIF file)]

Lung vascular permeability. There were no differences in extravascular plasma equivalents between the rabbits instilled with HCl alone and the rabbits given the control antibody. The extravascular accumulation of plasma equivalents in the lungs of the CD18-pretreatment groups was 70% lower than in both the positive control and the control antibody groups at 6 h (Fig. 3). In the NIF (anti-CD11b,18)-pretreatment group, the extravascular accumulation of plasma was significantly less (Fig. 3). In the neutrophil-depleted group, the extravascular accumulation of plasma equivalents was not different from that measured in the CD18- and NIF (CD11b,18)-pretreatment groups (Fig. 3).
Fig. 3. Endothelial permeability in lung measured as accumulation of vascular protein tracer 131I-albumin in extravascular spaces of lung and expressed as extravascular plasma equivalents in positive control, control antibody, CD18-pretreatment, PMN-depletion, NIF (CD11b,18)-pretreatment, and negative control groups at 6 h. Values are means and SD. Extravascular plasma equivalents were decreased by 70% in CD18-pretreatment and by 30% in NIF (CD11b,18)-pretreatment groups compared with positive control group and control antibody group at 6 h. * P < 0.05 vs. positive control group. dagger  P < 0.05 vs. negative control group. Dagger  P < 0.05 vs. control antibody group.
[View Larger Version of this Image (17K GIF file)]

Systemic blood pressure, heart rate, and peak airway pressure. No differences were observed in the blood pressure or heart rate at any time among the experimental groups (Table 1). The peak airway pressure rose in all groups within 5 min after instillation. Whereas the airway pressure in the positive control and the control antibody groups remained high, the airway pressure in the negative control group decreased by 6 h. In the pretreatment and neutrophil-depleted groups, the airway pressure tended to decrease, although this change did not reach statistical significance (Table 1).

Cell counts in bronchoalveolar lavage fluid and in peripheral blood. The number of polymorphonuclear leukocytes (PMN) lavaged from the air spaces in the acid-instilled group was 4-5 times higher than in controls (Fig. 4, Table 2). The CD18-pretreatment group count was >80% higher than in the positive control and the control antibody groups (Fig. 4, Table 2). Also, in the NIF (CD11b,18)-pretreatment group, the number of lavaged neutrophils was increased (>100%) (Fig. 4, Table 2). In the neutrophil-depleted group, no neutrophils were lavaged from the air spaces in any rabbit (Fig. 4). No significant differences were seen in the number of alveolar macrophages among the different groups (data not shown). In peripheral blood, the neutrophil count in the CD18-pretreatment group increased significantly by fourfold (from baseline 2,800 ± 1,900 to 8,100 ± 3,600 cells/µl at 6 h) and in the NIF (CD11b,18) group significantly by threefold (from baseline 2,000 ± 700 to 6,200 ± 1,300 cells/µl at 6 h). A significant twofold increase from baseline levels of 2,000 ± 1,200 to 4,600 ± 2,100 cells/µl was observed in the rabbits instilled with HCl alone and treated with the control antibody. In the neutrophil-depleted group, no circulating neutrophils were observed at any time during the experiment.
Fig. 4. Number of neutrophils lavaged from air spaces of rabbits in positive control, control antibody, CD18-pretreatment, PMN-depletion, NIF (CD11b,18) pretreatment, and negative control groups at 6 h. Values are means and SD. Number of PMN was 50 and 100% higher, respectively, in CD18- and NIF (CD11b,18)-pretreatment groups than in positive control, control antibody, and negative control groups at 6 h. * P < 0.05 vs. positive control group. dagger  P < 0.05 vs. negative control group. Dagger  P < 0.05 vs. control antibody group.
[View Larger Version of this Image (17K GIF file)]

Table  2.   White blood cells and neutrophils in lung as assessed by bronchoalveolar lavage 6 h after instillation of 1/3 normal saline or HCl, pH = 1.5 in 1/3 normal saline, in anesthetized, ventilated rabbits
Condition n White Blood Cells in Lavage, ×106 Neutrophils in Lavage, 106
HCl, pH = 1.5  7 75 ± 29dagger 46 ± 17dagger
HCl, pH = 1.5 + pretreatment with a nonspecific MAb 3 90 ± 19  55 ± 20 
HCl, pH = 1.5 + pretreatment with MAb CD11, CD18 5 168 ± 49*, dagger 131 ± 33*, dagger
HCl, pH = 1.5 + pretreatment with NIF, 10 mg · kg-1 · h-1 4 186 ± 76*, dagger 156 ± 81*, dagger
HCl, pH = 1.5 + neutrophil depletion 4 8 ± 5*, dagger 0 ± 0dagger
Control, 1/3 normal saline 4 43 ± 20* 15 ± 9*
Values are means ± SD; n = no. of rabbits. * P < 0.05, ANOVA vs. HCl alone, 6 h. dagger P < 0.05, ANOVA vs. control.

DISCUSSION

The results of this study confirm the hypothesis that neutrophil accumulation in the alveoli after acid aspiration is independent of CD18. However, pretreatment with an anti-CD18 monoclonal antibody and NIF anti-CD11b,18 led to a significant reduction in the severity of the acute lung injury from acid instillation. After neutralization of binding to CD18, acid-induced abnormalities in gas exchange, extravascular lung water, and lung vascular permeability were nearly completely prevented, even though the number of neutrophils lavaged from the air spaces was even higher than in acid-injured animals.

The three separate indexes of lung injury that were studied demonstrated internally consistent and convincing results. First, the alveolar-arterial O2 tension difference was nearly normal in acid-instilled rabbits pretreated with the anti-CD18 monoclonal antibody, indicating the absence of alveolar edema. Second, when CD18 binding was neutralized, extravascular lung water in acid-instilled rabbits was not different from that in rabbits instilled with saline alone. The water-to-dry weight ratios of 4.6-5.5 g H2O/g dry lung in CD18-pretreatment, NIF-pretreatment, neutrophil-depletion, and saline-instilled control groups are most consistent with mild interstitial edema. In the positive control and control antibody groups, on the other hand, the water-to-dry weight ratio was 7.6-7.7 g H2O/g dry lung; this quantity of extravascular lung water indicates significant alveolar edema. These differences become more obvious when the lung water is expressed as calculated milliliters of water accumulated in the lung in excess of that in a normal rabbit lung (3.2 g H2O/g dry lung). In the positive control and the control antibody groups, the excess H2O in both lungs was ~11.2-11.5 ml, or two- to sevenfold higher than the amount (1.5-5.0 ml) in the negative control, neutrophil-depleted, CD18-pretreatment, and NIF-pretreatment groups. Finally, the endothelial barrier was significantly protected in the acid-instilled rabbits given the anti-CD18 monoclonal antibody (Fig. 3). There was still a small increase in lung endothelial permeability that was not prevented by pretreatment or treatment with the monoclonal antibody to CD18. However, this mild increase in endothelial permeability was not associated with a significant increase in extravascular lung water. A small increase in lung endothelial permeability without an accompanying increase in extravascular lung water has previously been described in sheep given an endotoxin infusion (38).

The neutrophil mediates much of the acid-induced injury (9), but anti-CD18 did not decrease the influx of the neutrophils into the alveolar spaces. Therefore, we speculate that the CD18 antibody reduced the activation of PMN. Nathan and Sanchez (31) reported that the ability of the adherent neutrophil to undergo a respiratory burst in the presence of TNF-alpha (a major cytokine released in acid aspiration) (15) could be inhibited by anti-CD18 monoclonal antibody therapy. Anti-CD18 therapy prevented the decline in cAMP that was necessary for the neutrophil to prolong its oxidative burst and remain activated. Therefore, it is possible that, in this study, anti-CD18 treatment caused cAMP to remain elevated in the neutrophils. If this were true, then the neutrophils were less activated, even though they were still able to adhere and migrate into the lungs by CD18-independent mechanisms, as reported before (7). NIF (CD11b,18) has also been demonstrated to inhibit phorbol 12-myristate 13-acetate-induced neutrophil adhesion to fibrinogen, an event known to be CD11b,18 dependent (1). In the same study, NIF (CD11b,18) also was shown to inhibit neutrophil-dependent lung vascular injury by inhibiting neutrophil adhesion to the TNF-alpha -activated endothelium.

Interestingly, results similar to those in this study were reported by Goldman et al. (13) in a recent study of experimental acid aspiration. In that study, they found that the neutrophil receptor CD18 mediated remote, but not local, acid aspiration-induced acute lung injury. The data showed that the anti-CD18 monoclonal antibody had no effect on neutrophil accumulation in the aspirated segment, which was similar to the observations in this study. They also reported reduced extravascular lung water and less pulmonary edema, an effect that was observed in our study. Also, similar elevated neutrophil numbers have been observed after treatment with this monoclonal antibody in the rabbit ear ischemia-reperfusion model (23).

Why were the numbers of neutrophils in the bronchoalveolar lavage fluid of the CD18 and anti-CD11b,18 (NIF) groups (Fig. 4) significantly higher than in the rabbits instilled with HCl alone? One reason for the elevated neutrophil number in the lung after the anti-CD18 or the anti-CD11b,18 (NIF) pretreatment may have been that the preparations contained trace amounts of endotoxin that were not detected by the Limulus assay. However, a more likely cause is that anti-CD18 antibody therapy or anti-CD11b,18 (NIF) therapy did not prevent the release of chemotactic factors and, therefore, the stimulus for neutrophil entry into the alveoli would still be present. Because treatment with the anti-CD18 monoclonal antibody or the anti-CD11b,18 (NIF) resulted in a significant increase in circulating neutrophils, more neutrophils would have circulated through the lungs, potentially resulting in more alveolar neutrophils. Also, because similar observations were made with treatment by this antibody in a rabbit model of ear ischemia-reperfusion, the latter explanation is more likely. In that model, Lee et al. (23) demonstrated that even though the numbers of extravascular neutrophils were increased after the anti-CD18 treatment, the injury was dramatically attenuated, similar to our findings in this study. However, this may not have been the only explanation, because a small increase in circulating neutrophils was observed in the rabbits instilled with acid alone, although the increase was greater when the monoclonal anti-CD18 antibody or the anti-CD11b,18 (NIF) was administered. Another possibility is that acid aspiration might induce apoptosis of the alveolar neutrophils. It has recently been suggested that factors released in the lungs from patients with ARDS may modify the rate at which neutrophils become apoptotic (28). Another explanation may be related to the findings in a study on neutrophil migration after intradermal N-formyl-methionyl-leucyl-phenylalanine administration, in which it was demonstrated that the migration was independent of CD11b,18, whereas inhibition of CD11a and CD11c blocked neutrophil migration (35). Therefore, it is possible that neutrophil migration after acid aspiration is independent of CD11b inhibition and depends on CD11a and/or CD11c, but the development of injury depends on CD11b stimulation. Thus the results may indicate that blocking CD11b alone may not be sufficient to eliminate the vascular injury after acid aspiration.

The reason for the lower efficiency of NIF (CD11b,18) in protecting the lung from acid injury may be related to its ability to reduce or inhibit neutrophil activation. Nathan et al. (32) reported that anti-CD11b monoclonal antibodies do not inhibit the cytokine-induced respiratory burst of neutrophils, suggesting that NIF (CD11b,18) may work independently of neutrophils in this model of lung injury. Also, anti-CD11b monoclonal antibodies have been demonstrated to be ineffective as an inhibitor of rabbit neutrophil adhesion to endothelial cells and neutrophil migration in the skin (35). However, NIF (CD11b,18) has been demonstrated to inhibit both phorbol 12-myristate 13-acetate-induced neutrophil adhesion to fibrinogen and neutrophil-dependent lung vascular injury leading to edema formation after TNF-alpha administration (1). Again, inhibition of CD11b alone may not be sufficient to inhibit the neutrophil-dependent injury after acid aspiration.

In summary, we have found that either anti-CD18 or anti-CD11b,18 (NIF) pretreatment markedly reduced the severity of acute lung injury after acid instillation in rabbits, even though the number of neutrophils that were lavaged from the air spaces was even higher than in the sham antibody-treated or acid-instilled rabbits. Thus anti-CD18 treatment, as shown previously in vitro (31), can probably reduce acute lung injury in vivo by reducing the magnitude of neutrophil activation. These data confirm prior studies indicating that the presence of the neutrophils alone in the air spaces is not sufficient to cause acute lung injury (25, 26, 38).

Despite the paradoxical results regarding the elevated alveolar neutrophil counts and the lower extent of lung injury, there are reasons to be cautiously optimistic about new therapeutic approaches that inhibit neutrophil adhesion, chemotaxis, and activation for this form of acute lung injury (27). Acid aspiration-induced acute lung injury is one cause of ARDS for which there is often a clear-cut time of onset. Therefore, it may be more amenable to early therapeutic intervention. These experimental studies may provide a rational basis for the testing of novel therapeutic strategies in clinical studies of acid aspiration-induced acute lung injury.

ACKNOWLEDGEMENTS

The authors thank Oscar Osorio for valuable help with the surgical preparations of the animals and Minh Lam for the work with the analyses of the hemoglobin and total protein.

FOOTNOTES

   This work was supported by National Heart, Lung, and Blood Institute Grant HL-51854. H. G. Folkesson was supported in part by a grant from the American Lung Association of California Research Program.

Address for reprint requests: M. A. Matthay, Cardiovascular Research Institute, Univ. of California, 505 Parnassus Ave., HSW-1346, Box 0130, San Francisco, CA. 94143-0130.

Received 11 July 1996; accepted in final form 3 February 1997.

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