A potent inhibitor of cytosolic phospholipase A2, arachidonyl trifluoromethyl ketone, attenuates LPS-induced lung injury in mice

Takahide Nagase1, Naonori Uozumi2, Tomoko Aoki-Nagase1, Kan Terawaki2,3, Satoshi Ishii2, Tetsuji Tomita1, Hiroshi Yamamoto1, Kohei Hashizume3, Yasuyoshi Ouchi1, and Takao Shimizu2,4

Departments of 1 Geriatric Medicine, 2 Biochemistry and Molecular Biology, and 3 Pediatric Surgery, Graduate School of Medicine, University of Tokyo, and 4 Core Research for Evolutional Science and Technology of Japan Science and Technology Corporation, Tokyo 113, Japan


    ABSTRACT
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Acute respiratory distress syndrome (ARDS) is an acute lung injury of high mortality rate, and sepsis syndrome is one of the most frequent causes of ARDS. Metabolites of arachidonic acid, including thromboxanes and leukotrienes, are proinflammatory mediators and potentially involved in the development of ARDS. A key enzyme for the production of these inflammatory mediators is cytosolic phospholipase A2 (cPLA2). Recently, it has been reported that arachidonyl trifluoromethyl ketone (ATK) is a potent inhibitor of cPLA2. In the present study, we hypothesized that pharmacological intervention of cPLA2 could affect acute lung injury. To test this hypothesis, we examined the effects of ATK in a murine model of acute lung injury induced by septic syndrome. The treatment with ATK significantly attenuated lung injury, polymorphonuclear neutrophil sequestration, and deterioration of gas exchange caused by lipopolysaccharide and zymosan administration. The current observations suggest that pharmacological intervention of cPLA2 could be a novel therapeutic approach to acute lung injury caused by sepsis syndrome.

acute respiratory distress syndrome; lipopolysaccharide; sepsis; eicosanoid; leukotriene


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ACUTE RESPIRATORY DISTRESS SYNDROME (ARDS) is characterized by acute lung injury, and severe sepsis is one of the most important causes of ARDS (9, 10, 31). Although patients with ARDS are intensively treated with currently available drugs, the mortality rate for ARDS remains high, and it ranges from 40 to 70%. Potential mechanisms that cause ARDS include damage to the alveolar-capillary membrane and polymorphonuclear neutrophil (PMN) adhesion, activation, and sequestration, leading to respiratory failure (9, 10, 31).

Platelet-activating factor (PAF) and metabolites of arachidonic acid are potentially involved in the development of ARDS (23, 30, 32). PAF is a proinflammatory mediator produced from phospholipids (12-14). Thromboxanes (TXs) and leukotrienes (LTs) are potent mediators generated from arachidonic acid by cyclooxygenase and 5-lipoxygenase (7), respectively. TXA2 may increase lung permeability, whereas LTB4 is a potent neutrophil chemoattractant. Phospholipase A2 (PLA2) is a key enzyme for the production of proinflammatory mediators, including eicosanoids and PAF. Although a number of distinct types of PLA2 have been reported to be characterized, cytosolic PLA2 (cPLA2) is thought to be particularly important (29, 30, 34, 36, 37). The cPLA2 preferentially hydrolyzes phospholipids containing arachidonic acid and is activated by submicromolar concentration of Ca2+ and by phosphorylation of a serine residue (5, 16, 18, 33). Recently, it has been reported that an analog of arachidonic acid in which the -COOH functionality is replaced by -COCF3, named arachidonyl trifluoromethyl ketone (ATK), is a potent and selective slow-binding inhibitor of cPLA2 (33, 35).

In the present study, we hypothesized that pharmacological intervention of cPLA2 could affect acute lung injury. To test this hypothesis, we chose to use ATK as an inhibitor of cPLA2 and examined the effects of ATK in a murine model of acute lung injury induced by lipopolysaccharide (LPS) and zymosan administration.


    METHODS
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INTRODUCTION
METHODS
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Animal preparation. We used male C57BL/6 mice (7-8 wk old). Animals were anesthetized with pentobarbital sodium (25 mg/kg ip) and ketamine hydrochloride (25 mg/kg ip) in combination and then paralyzed with pancuronium bromide (0.3 mg/kg ip). Anesthesia and paralysis were maintained by supplemental administration of 10% of the initial dose every hour. After tracheostomy, a metal endotracheal tube (inside diameter 1 mm, length 8 mm) was inserted in the trachea. Animals were mechanically ventilated (model 683; Harvard Apparatus, South Natick, MA) with tidal volumes of 10 ml/kg and frequencies of 2.5 Hz. We opened the thorax widely by means of midline sternotomy and applied a positive end expiratory pressure of 2 cmH2O by placing the expired line underwater. During the experiments, oxygen gas was continuously supplied to the ventilatory system (FIO2 = 1.0). A heating pad was used to maintain the body temperature of animals. To assess the development of lung injury physiologically, we measured lung elastance (EL) and resistance (RL) as previously described (1, 21-27). Briefly, we measured the tracheal pressure (Ptr), flow, and volume (V). We calculated EL and RL by adjusting the equation of motion: Ptr = EL · V + RL(dV/dt) + K, where K is a constant. Changes in EL and RL reflect lung parenchymal alterations and stiffening of the lungs.

Experimental acute lung injury induced by LPS/zymosan administration. One minute before intravenous administration, two deep inhalations (three times tidal volume) were delivered to standardize volume history and measurements were made as baseline. In the physiological study, mice were divided into four experimental groups, i.e., saline-treated (n = 6), ATK/saline-treated (n = 4), LPS/zymosan-treated (n = 7), and ATK/LPS/zymosan-treated groups (n = 5). In the LPS/zymosan-treated group, mice received 3 mg/kg LPS from Escherichia coli O111:B4 (Sigma Chemical, St. Louis, MO) intravenously. Two hours later, 10 mg/kg of zymosan A from Saccharomyces cerevisiae (Sigma) were intravenously administered (19, 30). In the saline-treated group, animals received saline instead of LPS and zymosan in the same manner and served as controls. In the ATK-treated group, 20 mg/kg ATK (Cayman Chemical, Ann Arbor, MI) were administered intraperitoneally 30 min before saline or LPS administration. The current dose of ATK and timing of ATK administration were applied on the basis of previous reports (11, 20) and our preliminary experiments. In all groups, measurements were made at 30-min intervals for 4 h.

Assessment of respiratory failure. At the end of experiment, blood samples for gas analysis were obtained from the left ventricle. We then measured PaO2, PaCO2, and pH to assess the extent of respiratory failure (blood gas analyzer; AVL Medical Systems, Schaffhausen, Switzerland).

Bronchoalveolar lavage fluid. At the end of the experiment, bronchoalveolar lavage (BAL) was performed (1 ml of phosphate-buffered saline five times) in saline-treated (n = 6), LPS/zymosan-treated (n = 7), and ATK/LPS/zymosan-treated groups (n = 5). In each animal, 90% (4.5 ml) of the total injected volume was consistently recovered. After BAL fluid (BALF) was centrifuged at 450 g for 10 min, total and differential cell counts of the BALF were determined from the cell fraction (29, 30). The supernatant was stored at -70°C until measurement of protein was performed. The concentration of protein was measured by Lowry's method with bovine serum albumin as a standard.

TX and LT assay. TXA2 (measured as TXB2), LTB4, and LTC4/D4/E4 in the BALF were determined by enzyme immunoassay (EIA) kits (Amersham Pharmacia Biotech, Piscataway, NJ). The detection limits of the EIA assays for TXB2, LTB4, and LTC4/D4/E4 were 3.6, 6, and 10 pg/ml, respectively.

Myeloperoxidase activity assay. At the end of experiments, the left lungs were removed from mice of each group (n = 4, respectively). Myeloperoxidase (MPO) activity was measured as previously reported (3, 15). Briefly, the frozen lungs were weighed and homogenized in hexadecyltrimethylammonium bromide (HTAB) buffer (0.5% HTAB in 50 mM phosphate buffer, pH 6.0). The homogenates were sonicated and centrifuged at 40,000 g for 15 min. The supernatant was mixed with assay buffer containing potassium phosphate buffer, H2O2, and o-dianisidine hydrochloride. Then, the supernatant was placed in a spectrophotometer for reading at 460 nm as previously described (3, 15).

Histological study. At the end of experiments, the right lungs of the mice were removed and fixed with 10% formalin. After fixation, the tissue blocks obtained from midsagittal slices of the lungs were embedded in paraffin. Blocks were cut 4 µm thick with a microtome, and then hematoxylin-eosin staining was performed.

Data analysis. Comparisons of data among each experimental group were carried out with analysis of variance. If statistical significances were detected, a Scheffé test was then applied as a post hoc test. Data are expressed as means ± SE. P values <0.05 were taken as significant.


    RESULTS
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Physiological data following LPS/zymosan or saline administration. There were no significant differences in baseline EL and RL among each group. Fig. 1 and Table 1 demonstrate the physiological data after LPS/zymosan or saline administration. As shown, EL and RL in LPS/zymosan-treated group were significantly increased compared with saline-treated group, which reflects physiological alterations in lung parenchyma. The administration of ATK significantly reduced LPS/zymosan-induced responses in EL and RL, whereas there were significant differences between saline-treated and ATK/LPS/zymosan-treated groups.


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Fig. 1.   The time course of response in lung elastance in saline-treated (SAL, n = 6), arachidonyl trifluoromethyl ketone (ATK)/saline-treated (SAL+ATK, n = 4), LPS/zymosan-treated (LPS/Z, n = 7), and ATK/LPS/zymosan-treated groups (LPS/Z+ATK, n = 5). In LPS/zymosan-treated groups, zymosan was administered 2 h after LPS treatment, whereas saline was treated in the same fashion in the saline-treated groups. *P < 0.001 vs. saline-treated group; #P < 0.001 vs. LPS/zymosan-treated group.


                              
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Table 1.   Physiological results

Administration of LPS/zymosan elicited respiratory failure, which was not observed in saline-treated groups. Hypoxemia was prominent in LPS/zymosan-treated mice, whereas ATK administration reduced LPS/zymosan-induced hypoxemia (Fig. 2). After LPS/zymosan treatment, increases in PaCO2 and decreases in pH were observed, although there were no differences in PaCO2 or pH levels between saline-treated and ATK/LPS/zymosan-treated groups. As shown, ATK had little effect on physiological data in saline-treated groups.


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Fig. 2.   Effects of cytosolic phospholipase A2 (cPLA2) inhibitor ATK in hypoxemia induced by LPS/zymosan treatment. *P < 0.001 vs. saline-treated group; #P < 0.001 vs. LPS/zymosan-treated group.

Analyses of BALF. Table 2 and Figs. 3 and 4 summarize the analyzed data of BALF. As shown, LPS/zymosan administration increased protein amount and number of PMN in BALF, indicating LPS/zymosan induced protein leakage and PMN infiltration. The protein leakage and PMN sequestration were significantly attenuated by the treatment of ATK. Meanwhile, there were significant differences in BALF protein amount and number of PMN between saline-treated and ATK/LPS/zymosan-treated groups.

                              
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Table 2.   Total cell counts and cell fractions in BALF



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Fig. 3.   Effects of cPLA2 inhibitor ATK in protein leakage induced by LPS/zymosan treatment. BALF, bronchoalveolar lavage fluid. *P < 0.01 vs. saline-treated group; #P < 0.01 vs. LPS/zymosan-treated group.



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Fig. 4.   Effects of cPLA2 inhibitor ATK in neutrophil infiltration induced by LPS/zymosan treatment. PMN, polymorphonuclear neutrophil. *P < 0.001 vs. saline-treated group; #P < 0.001 vs. LPS/zymosan-treated group.

TX and LT assay. To assess the biosynthesis of cPLA2 products, we performed TXA2 (measured as TXB2), LTB4, and LTC4/D4/E4 assay of the BALF. Figures 5-7 summarize the results of BALF TXB2, LTB4, and LTC4/D4/E4 assay in each experimental group. LPS/zymosan administration markedly increased TXB2, LTB4, and LTC4/D4/E4 levels in BALF compared with the saline-treated group, whereas the levels of these eicosanoids were significantly reduced in the ATK/LPS/zymosan-treated group. However, there were significant differences in BALF TXB2, LTB4, and LTC4/D4/E4 levels between saline-treated and ATK/LPS/zymosan-treated groups.


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Fig. 5.   Effects of cPLA2 inhibitor ATK in thromboxane (TX) B2 production induced by LPS/zymosan treatment. *P < 0.05 vs. saline-treated group; #P < 0.01 vs. LPS/zymosan-treated group.



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Fig. 6.   Effects of cPLA2 inhibitor ATK in LTB4 production induced by LPS/zymosan treatment. *P < 0.001 vs. saline-treated group; #P < 0.05 vs. LPS/zymosan-treated group.



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Fig. 7.   Effects of cPLA2 inhibitor ATK in leukotriene (LT) C4/D4/E4 production induced by LPS/zymosan treatment. *P < 0.001 vs. saline-treated group; #P < 0.05 vs. LPS/zymosan-treated group.

MPO activity assay. To assess the PMN infiltration in the lung, we performed MPO activity assay. Figure 8 shows the results of MPO activity in lung tissue. LPS/zymosan administration markedly increased MPO activity in lungs compared with the saline-treated group, whereas the MPO activity was significantly attenuated in the ATK/LPS/zymosan-treated group. However, no significant difference in lung MPO activity was observed between saline-treated and ATK/LPS/zymosan-treated groups.


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Fig. 8.   Effects of cPLA2 inhibitor ATK in lung myeloperoxidase (MPO) activity induced by LPS/zymosan treatment (n = 4 for each group). *P < 0.05 vs. saline-treated group; #P < 0.05 vs. LPS/zymosan-treated group.

Histological study. Figure 9 represents lung histology following LPS/zymosan administration. As shown, LPS/zymosan administration induced prominent lesions, as well as alveolar thickening, distortion, and cellular infiltration. In contrast, the alveolar architecture is well preserved and histological changes are minimal in ATK-treated animals.


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Fig. 9.   Photomicrograph of lung tissues from LPS/zymosan-treated (A, C), and ATK/LPS/zymosan-treated (B, D) mice 4 h after LPS administration. Hematoxylin-eosin stain. Scale bar in A represents 200 µm in A and B and 50 µm in C and D.


    DISCUSSION
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INTRODUCTION
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The results of the current study show that cPLA2 is important in the pathogenesis of acute lung injury. Inhibition of cPLA2 significantly attenuated acute lung injury induced by endotoxemia. These observations indicate that pharmacological inhibition of cPLA2 may be an effective treatment for acute lung injury, probably because it inhibits production of inflammatory mediators including TXs and LTs.

The sepsis syndrome is the most frequent cause of ARDS and is associated with 35-45% incidence of ARDS development (9, 10). It is postulated that both endotoxemia and phagocytosis of bacteria are involved in the pathogenesis of ARDS associated with septic syndrome (6). Therefore, we used the current model of acute lung injury induced by combined administration of LPS and zymosan (19). In this model, circulating LPS and phagocytosis of bacterial particles by LPS-primed PMN elicit acute lung injury, which may mimic sepsis-associated acute lung injury.

After LPS/zymosan administration, we observed increases in EL, protein leakage, and PMN infiltration and severe exacerbation of gas exchange. PMN infiltration in the lung was confirmed by MPO activity assay and histology. Consistently, marked increases in TXs and LTs were detected in the BALF. These findings were significantly attenuated by the treatment of cPLA2 inhibitor ATK. Potential mechanisms by which cPLA2 mediates sepsis-induced acute lung injury include the release of proinflammatory mediators. The present results also suggest that the major mediator of PMN infiltration is a cPLA2 product, most probably LTB4 (38). Recent evidence using lung injury models overexpressing the LTB4 receptor shows that LTB4 is an important mediator of neutrophil-mediated lung injury (4). It is suggested that not only infiltration but also activation of PMN in lungs may be essential to induce the development of acute lung injury. The cPLA2-initiated pathways may mediate both infiltration and activation of PMN triggered by septic syndrome, resulting in sepsis-associated ARDS. In human neutrophils during sepsis, elevated cPLA2 expression and activity have been recently reported, suggesting that cPLA2 plays a major role in neutrophil function in septic syndrome (17).

Of note, it has been recently shown that acute lung injury induced by LPS/zymosan administration is attenuated in cPLA2 gene-disrupted mice (30). It seems that the effects of ATK administration are similar to those of cPLA2 gene disruption in terms of inhibiting lung injury. This observation may further confirm that the intervention of cPLA2 could be an effective approach to treat acute lung injury. However, differences were also found between these two studies. In this study, we measured TXB2, LTB4, and cysteinyl LTs (LTC4/D4/E4) in BALF to confirm the generation of cPLA2 products. Although the ATK administration significantly attenuated LPS/zymosan-induced production of TXB2, LTB4, and cysteinyl LTs, the ATK administration reduced each eicosanoid by 73, 47, and 27%, respectively, compared with LPS/zymosan administration. In contrast, cPLA2 gene disruption reduced each eicosanoid by >90% in this model, compared with LPS/zymosan administration in wild-type mice. This finding suggests that the present manner of ATK administration may still be insufficient to inhibit cPLA2 completely. Because it is postulated that pharmacological intervention of cPLA2 could be useful in the management of ARDS, the development of novel cPLA2 inhibitors warrants future research.

In the present model of acute lung injury, we observed that the levels of PaCO2 and pH in the ATK/LPS/zymosan-treated group were the same as in saline-treated controls. However, LPS/zymosan-induced increases in EL, severity of hypoxia, BALF protein, PMN, and eicosanoids were significantly attenuated but not eliminated by the treatment of ATK. These observations indicate that factors other than cPLA2 may also play a role and contribute to physiological alteration. Recently, it has been demonstrated that secretory PLA2 (sPLA2), the other type of PLA2, mediates LPS-induced lung injury and that the inhibition of sPLA2 may also represent a therapeutic approach to acute lung injury (2). In addition, it has been suggested that oxygen radicals, adhesion molecules, and cytokines are also involved in this mechanism (8, 28). Recently, it was reported that cPLA2 activation is essential for integrin-dependent adhesion of leukocytes (39). If one considers that there are as yet no pharmacological agents to reverse pulmonary edema and increase survival rates, these factors are potential targets to develop agents. The current study suggests that the intervention of cPLA2 could be a promising clue to improve management of ARDS.

In summary, the inhibition of cPLA2 significantly attenuated lung damage and respiratory failure induced by LPS/zymosan treatment. The current observations suggest that cPLA2 products are involved in the pathogenesis of acute lung injury caused by septic syndrome. Inhibition of cPLA2-initiated pathways might provide a novel and potential therapeutic approach to ARDS, to which no pharmaceutical agents are currently available.


    ACKNOWLEDGEMENTS

This work was supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan and grants-in-aid for Comprehensive Research on Aging and Health from the Ministry of Health, Labour and Welfare, Japan, a grant from the Mochida Memorial Foundation for Medical and Pharmaceutical Research, a grant from the Yamanouchi Foundation for Research on Metabolic Disorders, a grant from the Smoking Research Foundation, and a grant from the Novartis Foundation for Gerontological Research. T. Aoki-Nagase is a Research Resident of Japan Foundation for Aging and Health.


    FOOTNOTES

Address for reprint requests and other correspondence: T. Nagase, Dept. of Geriatric Medicine, Faculty of Medicine, Univ. of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan, 113-8655 (E-mail:takahide-tky{at}umin.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published December 27, 2002;10.1152/ajplung.00396.2002

Received 18 November 2002; accepted in final form 25 December 2002.


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Am J Physiol Lung Cell Mol Physiol 284(5):L720-L726
1040-0605/03 $5.00 Copyright © 2003 the American Physiological Society




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