INTRODUCTION

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Reexpansion pulmonary edema (REPE) is a rare, but sometimes fatal, complication combining pneumothorax, pleural effusion, and atelectasis. It has been proposed that REPE develops after rapid reexpansion of the collapsed lung (1). Although the precise pathogenesis of this disease is unclear, it has been suggested that mechanical stimuli (4), a decrease in surfactant (5), or released biochemical mediators (1, 6) induce an increase in pulmonary microvascular permeability, which results in pulmonary edema (1, 4, 7). Because pathological studies have revealed the intrapulmonary accumulation of neutrophils, and because lung edema fluid analysis has demonstrated increased neutrophil counts and/or polymorphonuclear leukocyte elastase levels (1, 6, 7), it is reasonable to speculate that neutrophils profoundly contribute to the development of this disorder.

There are several candidates as an inducer of neutrophil accumulation in this disease (1, 6). Among them, interleukin-8 (IL-8), a member of C-X-C chemokine, is a very potent chemotactic and activating factor for neutrophils and has been shown to recruit neutrophils into the extravascular spaces in the inflammatory phase of various diseases such as acute lung injury (8, 9). IL-8 is produced by a wide variety of cells in the lung, including alveolar macrophages (AMs), bronchial epithelial cells, and pulmonary fibroblasts (10). During the past several years, it has been established that IL-8 is produced in a large amount and plays an important role in various inflammatory lung diseases, including acute respiratory distress syndrome (ARDS) (9, 13, 14), inhalation injury (15), and idiopathic pulmonary fibrosis (12). Previously, we reported a case of REPE, in whom the IL-8 level in bronchoalveolar lavage fluid (BALF) was increased and paralleled the bronchoalveolar lavage (BAL) neutrophil counts during the course (1). In the present study, to elucidate the pathophysiological role of IL-8 in REPE, we systematically examined the expression of IL-8 and protective effect of a specific monoclonal anti-IL-8 antibody (Ab) on reexpansion lung injury in rabbits.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animal Preparations

Japanese white male rabbits weighing 1.1 ± 0.2 kg (mean ± SEM) were used (n = 58). These animals were free of respiratory tract infections and were housed under standard conditions in animal care facilities. The protocols were approved by the institutional review board for animal studies. Rabbits were anesthetized with an intramuscular injection of 100 mg/kg ketamine and 5 mg/kg xylazine. In the sham surgery (Sham) group, posterolateral thoracotomy was performed. In the collapsed (Collapse), reexpansion (Reex), anti-IL-8 Ab (IL-8 Ab), and control Ab (CTL Ab) groups, thoracotomy was performed and the left main bronchus was completely clamped with a clip. A string was tied with the clip and was exteriorized to enable the subsequent declamping of the left main bronchus. After wound closure, the animals were allowed to recover for 36 h before the initiation of the experiments.

Preparation of Monoclonal Ab to Rabbit Recombinant IL-8

The generation of the monoclonal Ab to rabbit recombinant IL-8 (rIL-8) (ARIL8.2; Genentech Inc., South San Francisco, CA) has previously been described in detail (16). ARIL8.2 had high abilities to recognize rabbit IL-8, to inhibit binding of ¹²⁵I-labeled rabbit rIL-8 to its receptor, to block rabbit rIL-8 induced signal transduction in neutrophils via its receptor, and to inhibit rabbit rIL-8-induced chemotactic activity for rabbit neutrophils (16, 17). ARIL8.2 had a high affinity for rabbit IL-8 (K_d = 0.42 nM). ARIL8.2 did cross-react with human IL-8, but not with closely related cytokines (human melanoma growth stimulating activity [hMGSA], platelet factor-4, -thromboglobulin), other human cytokines (IL-1, tumor necrosis factor- [TNF-]), or other chemotactic factors (formylmethionylleucylphenylalanine [fMLP], C5a). The antibody was sterilely filtered and endotoxin was confirmed to be undetectable by the Limulus assay.

Experimental Design

The protocol for the experiment is provided in Figure 1. On the day of experiments, 2 ml of 93 kBq/ml ¹²⁵I-albumin was injected intravenously in all groups, including Sham, Collapse, Reex, IL-8 Ab, and CTL Ab, to assess the pulmonary extravasated albumin. In the Reex, IL-8 Ab, and CTL Ab group, the left main bronchus was reopened by pulling on the string 10 min after ¹²⁵I-albumin injection. At this time, a small chest tube (9 French) was inserted (2 to 3 cm) through a left midintercostal space and continuous suction at 10 cm H₂O was initiated. In the IL-8 Ab group, 10 mg/kg of specific anti-IL-8 monoclonal Ab (ARIL8.2) and in the CTL Ab group, 10 mg/kg of nonimmunized Ab (no. 55939; Organon Teknika Corp., Durham, NC) were injected intravenously 60 min before reopening the left main bronchus. One hundred fifteen minutes after reexpanding the left lung, 2 ml of 93 kBq/ml ¹³¹I-albumin was injected intravenously to estimate contaminated blood in the lung specimens. After the injection, each animal received 2,000 IU of intravenous heparin. To prevent hypotension and dehydration, 25 ml/kg/h of normal saline was administered intravenously to each animal. 120 min after continuous suction, the animal was rapidly injected with 1 ml pentobarbital and was killed. The chest was opened, a blood sample was taken from the inferior vena cava, and the lungs were resected after clamping at the sites of both hila. Both lungs were drained of free blood by gently blotting on paper towels, and divided into five pieces, each of which was placed in a preweighed glass tube for the determination of the weight and radioactivity. The counts of both ¹²⁵I and ¹³¹I were examined for both lungs and blood specimens with a gamma counter (ARC-300; Aloka, Tokyo, Japan).

View larger version (22K): [in this window] [in a new window]   Figure 1.   Study design and experimental protocol. The experimental groups consist of: sham surgery (Sham), collapsed (Collapse), reexpansion (Reex), anti-IL-8 antibody (IL-8 Ab), and control antibody (CTL Ab) groups. Sham group: only thoracotomy was performed. Collapse group: left main bronchus was clamped for 38 h. Reex group: left main bronchus was declamped after 36 h of clamping and the collapsed lung was reexpanded for 2 h. IL-8 Ab group and CTL Ab group: either 10 mg/kg of specific anti-IL-8 antibody or a nonimmunized antibody was injected 60 min before reopening the left main bronchus. 125I- albumin was injected intravenously 10 min before reopening the left main bronchus to evaluate transvascular permeability. 131I-albumin was injected intravenously 5 min before sacrifice to correct blood contamination.

In several additional experiments, rabbits were examined following the same protocol except radioisotope injection to collect lung tissue and BALF for later analysis. The resected lung tissue was kept frozen at 80° C until analysis.

Assessment of Pulmonary Edema and Capillary Permeability

Transvascular albumin flux was assessed from the ratio of ¹²⁵I-albumin counts of the lung tissue to that of the plasma per unit weight (T/P ratio) in both right and left lungs. The lung ¹³¹I counts were subtracted from lung ¹²⁵I counts to correct the contaminated blood volume in the lung pieces. The mean value of the T/P ratio in five lung specimens was adopted as the indicator of pulmonary microvascular permeability of each lung field (18).

Pulmonary edema was assessed by the lung wet-to-dry weight (W/D) ratio in right and left lungs. After the wet weights of the lung tissues and those of 1 ml blood specimens were measured, they were completely dried up in a vacuum oven (DP22; Yamato Scientific, Tokyo, Japan) at 95° C and 20 cm H₂O for 48 h to remove any gravimetrically detectable water. The dry weights of the lung and blood specimens thus prepared were determined and the lung W/D ratio was calculated after correcting the contaminated blood volume estimated by the gamma count of ¹³¹I counts in each lung specimen. The mean W/D ratio calculated from five lung pieces in each lung was used as the measure of pulmonary extravascular water accumulation in the respective lung field (18).

BAL

The sequestration of inflammatory cells in the airspaces was assessed by BAL performed for the right and left lower lobes in all groups studied. Before BAL, blood on the surface of the lung was rinsed off with saline to minimize blood contamination. BAL was performed by injecting three times with 5 ml saline followed by gentle aspiration of the fluid from the lower lobe after securing the catheter located within the main bronchus of either right or left lung. No significant differences were observed in the amount of recovered BALF among the groups. The total cell numbers in BALF were counted with a hemocytometer. For differential counts of leukocytes in BALF, cytospin smear slides were prepared (Sakura-Seiki, Tokyo, Japan) by correcting the contamination of erythrocytes. The smear was stained with a modified Wright's solution (Diff-Quik; American Scientific Product, McGraw Park, IL). Differential cell counts were performed on 200 cells in the smear.

Enzyme-linked Immunosorbent Assay (ELISA) for IL-8

The lung tissue IL-8 concentrations in both right and left lungs were assessed in the Sham, Collapse, and Reex groups. Using a homogenizer (Kinematica AG, Luzern, Switzerland), lung tissue homogenate was prepared from 500 mg of frozen lung tissue, to which 2 ml of phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA; Sigma, St. Louis, MO) and 0.05% Tween 20 (Sigma) was added. The homogenate thus prepared was centrifuged at 3,000 rpm for 15 min at 4° C, and the supernatant was stored at 80° C until analysis. IL-8 levels were measured with a sandwich enzyme-linked immunosorbent assay (ELISA) (Genentech) as described previously (19). In this assay, 8C8. 11.17 was used as the primary capture monoclonal Ab and 4D4.1.15 was the secondary monoclonal Ab for recognition of the antigen. Optical density was measured with an ELISA plate reader (Sjeia II; Sanko Junyaku Co., Tokyo, Japan) at a wavelength of 450 nm. Absolute values of IL-8 concentration in the supernatant were determined on the basis of the standard curve constructed from the solution containing known quantities of IL-8.

Quantitation of IL-8 Messenger RNA (mRNA) by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)

Total RNA was extracted from each lung tissue using RNAeasy kits (Quiagen, Germany). Single-strand complementary DNA (cDNA) was synthesized using M-MLV reverse transcriptase (GIBCO BRL, Rockville, MD) and oligo(dT)₂₀ primer. To measure gene expression, we performed a newly developed quantitative PCR method using a dual-labeled fluorogenic probe and 7700 Prism sequence detector (Perkin Elmer, Foster City, CA) as proposed by Gibson and coworkers (20). The probe was designed between the forward and reverse primer sites, and was labeled with a reporter fluorescent dye (6-carboxyfluorescein [FAM]) at the 5'-end and a quencher fluorescent dye (6-carboxy-tetramethyl rhodamine [TAMRA]) at the 3'-end. Accumulation of PCR products was detected directly by monitoring the increase in fluorescence of the reporter dye.

Measurement of Myeloperoxidase (MPO) Activity

The lung tissue MPO activities in both right and left lungs were assayed to assess the numbers of recruited neutrophils to the lungs in the Sham, Collapse, and Reex groups. MPO activities in lung tissue homogenate were measured with the SUMILON peroxidase assay kit (ML-11300; Sumitomo Bakelite, Tokyo, Japan), according to the manufacturer's instructions.

Histopathological and Immunohistochemical Examinations

The lower lobes of both lungs were processed for histopathological and immunohistochemical examinations. The tissue slices, 3 mm in thickness, were fixed in 4% paraformaldehyde, and frozen in OCT compound (Miles Inc., Elkhart, IN) on dry ice and acetone. The histological sections were prepared with a cryostat, dried for 30 min, and stained with hematoxylin and eosin for microscopic examination. For immunohistochemistry, the sections incubated with 10% goat serum in PBS for 30 min were incubated with either mouse monoclonal Ab against IL-8 (ARIL8.2) or control nonimmunized IgG (Sigma) for 2 h at room temperature. Subsequently, the sections were incubated at room temperature for 30 min with goat anti-mouse immunoglobulins conjugated to peroxidase-labeled dextran polymer (no dilution; En Vision+, Peroxidase, Mouse; DAKO Corp., Carpinteria, CA). Color was developed with 3,3'-diaminobenzidine tetrahydrochloride (0.2 mg/ml; Dojindo Laboratories, Kumamoto, Japan) in 0.5 M Tris-HCl (pH 7.6) containing 0.003% hydrogen peroxide, and the sections were counterstained with methyl green (Merck, Darmstadt, Germany).

Statistical Analysis

All data are presented as mean ± SEM. A one-way analysis of variance (ANOVA) followed by Fisher's least significant difference (LSD) test was applied to detect statistically significant differences among groups. A paired t test was used to compare the values between right and left lungs. Significant differences were accepted when the value of p was less than 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Development of Lung Injury in Reexpanded Lung

Figure 2A shows the T/P ratio of the left lungs in the Sham, Collapse, Reex, IL-8 Ab, and CTL Ab groups (n = 5 for each group). Although the left lung T/P ratio was augmented in the Reex group in comparison with that in the Sham or Collapse group, it was conspicuously inhibited when treating the animal with anti-IL-8 Ab, but not with control Ab.

View larger version (17K): [in this window] [in a new window]   Figure 2.   Lung T/P ratio and lung W/D ratio. Values of the 125I-albumin concentration ratio of lung tissue to plasma (lung T/P ratio), a parameter of pulmonary microvascular permeability (A) and lung tissue wet/dry ratio (lung W/D ratio), a parameter of pulmonary edema (B) in the left lungs of the Sham, Collapse, Reex, IL-8 Ab, and CTL Ab groups. *p < 0.01 versus Sham, #p < 0.05 versus Collapse and IL-8 Ab, §p < 0.001 versus Sham, p < 0.005 versus Collapse and IL-8 Ab. +p < 0.05 versus Sham and Collapse. Values are mean ± SEM.

Figure 2B shows the W/D ratio of the left lungs in the Sham, Collapse, Reex, IL-8 Ab, and CTL Ab groups (n = 5 for each group). The W/D ratio of the left lungs was much higher in the Reex and CTL Ab groups than that of the Sham and Collapse groups. The mean W/D ratio of the IL-8 Ab group tended to be lower than the value obtained in the Reex or CTL Ab groups, though statistical significance was not attained.

The total and differential cell numbers in BALF of the left lungs are shown in Figure 3 (n = 5 for each group). There were no significant differences in total cell and macrophage numbers among the groups. The neutrophil numbers in the Reex group were increased as compared with those of the Sham and Collapse groups. These numbers were significantly decreased in the IL-8 Ab group, but not in the CTL Ab group. In addition, the lymphocyte numbers of the left lung were higher in the Reex group than the Sham group. The mean lymphocyte numbers in the Reex group were inclined to be higher than those of the Collapse and IL-8 Ab groups (p = 0.07 versus Collapse, p = 0.06 versus IL-8 Ab).

View larger version (38K): [in this window] [in a new window]   Figure 3.   Total cell and differential cell numbers in BALF of the left lungs in the Sham, Collapse, Reex, IL-8 Ab, and CTL Ab groups. *p < 0.0005 versus the Sham, Collapse, and IL-8 Ab. #p < 0.005 versus the Sham, Collapse, and IL-8 Ab. p < 0.05 versus the Sham. Values (×105/ml) are mean ± SEM.

The T/P and W/D ratios, and the cell counts in BALF estimated for the right lung did not differ among any groups studied and coincided with the values for the left lung in the Sham group.

IL-8 Levels and Their Expression in Reexpanded Lung

The IL-8 contents in the left lung tissue homogenate of the Sham, Collapse, and Reex groups (n = 5 for each group) are shown in Figure 4A. The left lung IL-8 concentrations in the Reex group were found to be distinctly higher than those in the Sham group. This tendency, however, was not observed between the Collapse and Sham groups. On the other hand, little difference was seen for the right lung IL-8 levels among the Sham, Collapse, and Reex groups. The right lung IL-8 concentrations in the three groups were statistically the same as those observed for the left lung in the Sham group, leading to approximately 1.6-fold enhancement in IL-8 production in the left lung compared with that in the right lung in the Reex group.

View larger version (15K): [in this window] [in a new window]   Figure 4.   Lung IL-8 concentrations and IL-8 mRNA expression. IL-8 levels (A) and IL-8 mRNA expression indicated by the ratio to that of GAPDH (B) in the left lung tissue homogenates of the Sham, Collapse, and Reex groups. *p < 0.05 versus the Sham. Values are mean ± SEM.

The expression of left lung IL-8 mRNA in the Sham, Collapse, and Reex groups is shown in Figure 4B (n = 5 for each group). Values are given as the ratio against reduced glyceraldehyde-phosphate dehydrogenase (GAPDH). In the Reex group, the IL-8 mRNA expression of the left lung was largely upregulated in comparison with that in the Sham group. On the other hand, a trend qualitatively similar to that of IL-8 contents was observed in the right lung IL-8 mRNA expression, i.e., there were no differences in IL-8 mRNA among the three groups, with the result that IL-8 mRNA expression in the left lung was approximately fourfold greater than that in the right lung in the Reex group.

MPO Activities in Reexpanded Lung

In the Reex group, the MPO activities of the left lung were significantly higher (1.36 ± 0.24) than those in the Sham group (0.64 ± 0.17, p = 0.04) and Collapse group (0.50 ± 0.27, p = 0.02) (n = 5 for each group). There were no differences in the MPO activities of the right lungs among the three groups. Furthermore, the MPO activities did not differ between the left and the right lungs in the Sham group.

Histopathological and Immunohistochemical Findings

The histopathological conditions of the left lungs in the Sham, Collapse, Reex, and IL-8 Ab groups are depicted in Figure 5 (n = 4 for each group). The histopathological findings in the Sham and Collapse groups were almost normal, whereas the Reex group revealed edematous alveolar septa in which significant sequestration of both mononuclear and polymorphonuclear leukocytes was observed. Moreover, both lung injury and leukocyte infiltration into alveolar septa were obviously attenuated in the IL-8 Ab group. There were little morphological abnormalities in the right lungs of the four groups.

View larger version (114K): [in this window] [in a new window]   Figure 5.   Histopathological examination. Histopathological findings of the left lungs stained with hematoxylin and eosin in the Sham (A), Collapse (B), Reex (C ), and IL-8 Ab groups (D). Bars represent 200 µm in length.

The immunohistochemical localization of IL-8 of the left lungs in the Sham, Collapse, Reex, and IL-8 Ab groups is depicted in Figure 6 (n = 4 for each group). In the Sham group, no IL-8 expression was demonstrated. In the Collapse group, the expression of IL-8 was localized to AMs, whereas it was clearly recognized in AMs and, to a lesser extent, in epithelial cells in the Reex group.

View larger version (99K): [in this window] [in a new window]   Figure 6.   Immunohistochemical examination. Immunohistochemical localization of IL-8 in the left lungs in the Sham (A), Collapse (B), and Reex groups (C ). Bars indicate 50 µm in length. Arrowhead: IL-8 positive AM. Arrow: IL-8 positive epithelial cell.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Importance of IL-8 in Development of REPE

REPE occurs when a chronically collapsed lung is rapidly reexpanded (1). Previous studies have demonstrated that this disease belongs to a category of noncardiogenic pulmonary edema, which occurs as a result of an increase in microvascular permeability (1, 4, 7). Up to the present, however, the mechanisms of the increase in microvascular permeability in REPE are largely unknown, because of its low incidence and the lack of adequate experimental models. Previous clinical and experimental studies indicated multiple factors inducing the pathophysiology of REPE, in which the final process was likely to be caused by increased pulmonary microvascular permeability. The important pathophysiological factors in relation to this disease have been believed to be rapid reexpansion and reperfusion of hypoxic lung tissue with oxygenated blood (2, 3). During unilateral pneumothorax, chronically collapsed lung is exposed to sustained hypoxia because of the decrease in ventilation/perfusion ratio of this lung. As a result of hypoxic vasoconstriction, the blood flow in the collapsed lung is expected to decrease. In fact, using ^99mTc-macroaggregated albumin, our preliminary study demonstrated that blood flow in the collapsed lung fell by half as much as that of the opposite lung (data not shown). During reexpansion of a collapsed lung, oxygen tension increases. Several studies indicated that this reoxygenation caused the large production of oxygen-derived free radicals including superoxides, that resulted in the increase of pulmonary microvascular permeability in the experimental reexpansion lung injury models (21).

Mechanical stimuli to the pulmonary vessels and alveoli during reexpansion have been proposed as another candidate for the triggering of REPE, although there is little experimental evidence (4). In addition, changes in pulmonary lymph flow, reduced lung surfactant volume (5), or bronchial occlusion have also been discussed as the possible cause of REPE. This disease usually occurred in the ipsilateral lung, but occasionally in the bilateral lungs (22). Thus, it is reasonable to speculate that humoral mediators are involved in the development of REPE. Various mediators have been reported to be important direct or indirect factors for this lung injury, such as thromboxane A₂, polymorphonuclear leukocyte elastase, -1-protease inhibitor, leukotriene B₄, and C5a (1, 6). Recently, it has been clarified that neutrophils and their products play major roles not only in defense against microbial infection, but also in tissue injury (23). In parallel with the above substances, the significance of neutrophil chemotactic mediators, including complement fragments, bioactive lipids, and cytokines, has been extensively addressed. Among them, IL-8 is one of the most potent chemoattractants for neutrophils (8) and several groups of investigators including us have shown its augmented production in various systemic and lung diseases, such as acute respiratory distress syndrome (ARDS) (9, 13, 14), inhalation injury (15), and acute exacerbation of idiopathic pulmonary fibrosis (12).

Based on these facts, we attempted, in the present study, to solve the issue of whether IL-8 plays a significant role in the development of REPE. We found that reexpansion of a collapsed lung enhanced IL-8 production through upregulation of IL-8 mRNA in the lung, which, in turn, induced inflammatory cell accumulation and conspicuous pulmonary edema in association with increased microvascular permeability (Figures 2, 3, and 4). The significantly increased MPO activities in the reexpanded lung also confirmed that the numbers of recruited neutrophil were increased in the reexpanded lung. Furthermore, we found that pretreatment with anti-IL-8 Ab inhibited the accumulation of inflammatory cells, the increase in microvascular permeability, and the extent of the lung injury (Figures 2, 3, and 5), leading to the tendency to reduce the pulmonary edema (Figure 2). These findings are highly consistent with the idea that augmented production of lung IL-8 plays an important role in developing and establishing pathophysiological aspects of REPE. IL-8 has been considered to function as an important chemotactic and activating factor for neutrophils as well as lymphocytes (8). This is clearly supported by the present study, in which the enhanced accumulation of neutrophils and lymphocytes in BALF during reexpansion of a collapsed lung was attenuated by treatment with anti IL-8 Ab (Figure 3). To the best of our knowledge, this may be the first report strongly indicating that the potent chemoattractant, IL-8, is a major contributing factor to the development of REPE.

Cellular Sources of IL-8 Production in REPE

The previous in vitro studies demonstrated that anoxic preconditioning followed by oxygen stress augmented production of monocyte-derived and polymorphonuclear leukocyte- derived IL-8 (24, 25). Two studies have indicated that severe hypoxia (1 to 3% oxygen)-reoxygenation modulated neutrophil migration on the epithelial cell layer with overproduction of IL-8 (26) and neutrophil adhesion to endothelial cell that was reduced by an antibody against IL-8 (27). In addition, Karakurum and coworkers (28) reported that a hypoxic condition (oxygen tension; 14 to 18 mm Hg) induced the expression and production of IL-8 from endothelial cells in vitro. They (28) also showed that a hypoxic condition (oxygen tension; 30 to 40 mm Hg) induced pulmonary leukostasis and increased the level of interferon gamma inducible protein-10 (IP-10), which is a murine homologue in the chemokine family related to human IL-8. Tamm and colleagues (29) showed that hypoxia (3% oxygen) enhanced transcription and translation of IL-8 and IL-6 in pulmonary fibroblasts and vascular smooth muscle cells. Utgaard and colleagues (30) suggested that IL-8 prestored in microvascular endothelial cells was secreted and activated neutrophil adhesion immediately after acute inflammation. In addition, alveolar epithelial cells were shown to release IL-8 under a stretch stress condition (31).

Summarizing the in vitro studies as described previously, IL-8 can be produced by a wide variety of cells, including monocytes, polymorphonuclear leukocytes, fibroblasts, endothelial cells, vascular smooth muscle cells, and epithelial cells under various insults. In the collapse-reexpansion lung model employed in the present study, however, IL-8 was positive chiefly in AMs and, to a lesser extent, in alveolar epithelial cells (Figure 6), suggesting that IL-8 would be produced predominantly by AMs and partially by alveolar epithelial cells, but not by other cells such as fibroblasts and endothelial cells, at least under a specific condition, in which the lung was initially collapsed and subsequently reexpanded.

Mechanisms of IL-8 Production

IL-8 mRNA expression and related production of IL-8 were augmented in the reexpanded lung but not in the collapsed lung (Figure 4). By combining the IL-8 quantitative data (Figure 4) in the lung homogenate with the immunohistochemical analysis (Figure 6), one can infer that IL-8 production has already started to increase during the collapsed phase of the lung, but it is much more noticeably augmented after reexpansion. If a method more sensitive than that applied in the present study can be used, subtle increases in IL-8 mRNA expression and IL-8 production are also expected to be detected in the collapsed lung. Sustained hypoxia during the collapse and some unknown causes during the reexpansion, including reoxygenation, may contribute to the enhancement of lung IL-8 production. Because hyperosmotic stress and stretching have been suggested to enhance IL-8 production in mononuclear cells and alveolar epithelial cells, respectively (31, 32), these mechanical factors may also be the candidates for a large increase in IL-8 mRNA expression and IL-8 production during reexpansion of the lung.

Clinical Applicability

We showed that pretreatment with an IL-8 neutralizing antibody is possibly applicable for the treatment of this disease (Figures 2, 3, and 5). Several reports in the literature indicated that treatment with anti-IL-8 antibody attenuated lung injury, such as immune complex-associated alveolitis, endotoxin pleurisy (16, 19), acute lung injury caused by acid aspiration (17), lung injury by cobra venom factor (33), and endotoxemia- induced lung injury (34). In the study of Sekido and coworkers (35), an anti-IL-8 monoclonal Ab prevented neutrophil infiltration and lung injury caused by ischemia-reperfusion in rabbits. The present experimental results indicated that REPE would also be ascribed to overproduction of IL-8 during collapse and subsequent reexpansion of the lung. Among these various IL-8-mediated lung diseases, the reexpansion lung injury is unique in that one can determine the time point of the insult, namely the collapse and reexpansion of the lung. The current results suggest that pretreatment with anti-IL-8 antibody may be a protective therapy for this disease, especially in the high-risk patients whose lungs are collapsed for a prolonged period.