Lung production of platelet-activating factor acetylhydrolase in oleic acid-induced acute lung injury

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Prostaglandins, Leukotrienes and Essential Fatty Acids 77 (2007) 1–8 www.elsevier.com/locate/plefa

Lung production of platelet-activating factor acetylhydrolase in oleic acid-induced acute lung injury$ Jorge I. Salluha,b,, Alexandre V. Pinoc, Adriana R. Silvaa, Rachel N. Gomesa, Heitor S. Souzad, Jose Roberto Lapa e Silvad, Frederico C. Jandrec, Antonio Giannella-Netoc, Guy A. Zimmermane, Diana M. Stafforinif, Stephen M. Prescottf, Hugo C. Castro-Faria-Netoa, Patrı´ cia T. Bozzaa, Fernando A. Bozzaa,g Immunopharmacology Laboratory, Department of Physiology and Pharmacodynamics, Fundac- a˜o Oswaldo Cruz, Rio de Janeiro, RJ, Brazil b Intensive Care Unit, Instituto Nacional de Caˆncer, Rio de Janeiro, RJ, Brazil c Pulmonary Engineering Laboratory, Department of Biomedical Engineering- COPPE- Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil d Multidisciplinary Laboratory, Hospital Universita´rio Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil e Program in Human Molecular Biology and Genetics, Department of Internal Medicine, University of Utah, Salt Lake City, UT, USA f Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, USA g ICU, Instituto de Pesquisas Clı´nicas Evandro Chagas, Fundac- a˜o Oswaldo Cruz, Rio de Janeiro, RJ, Brazil a

Received 11 December 2006; received in revised form 6 March 2007; accepted 3 April 2007

Abstract Platelet-activating factor (PAF) is a proinflammatory mediator that plays a central role in acute lung injury (ALI). PAFacetylhydrolases (PAF-AHs) terminate PAF’s signals and regulate inflammation. In this study, we describe the kinetics of plasma and bronchoalveolar lavage (BAL) PAF-AH in the early phase of ALI. Six pigs with oleic acid induced ALI and two healthy controls were studied. Plasma and BAL samples were collected every 2 h and immunohistochemical analysis of PAF-AH was performed in lung tissues. PAF-AH activity in BAL was increased at the end of the experiment (BAL PAF-AH Time 0 ¼ 0.00170.001 nmol/ml/min/g vs Time 6 ¼ 0.03170.018 nmol/ml/min/g, p ¼ 0.04) while plasma activity was not altered. We observed increased PAF-AH staining of macrophages and epithelial cells in the lungs of animals with ALI but not in healthy controls. Our data suggest that increases in PAF-AH levels are, in part, a result of alveolar production. PAF-AH may represent a modulatory strategy to counteract the excessive pro-inflammatory effects of PAF and PAF-like lipids in lung inflammation. r 2007 Elsevier Ltd. All rights reserved.

1. Introduction $

This work was performed at the Immunopharmacology Laboratory, Fundac- a˜o Oswaldo Cruz and Pulmonary Engineering Laboratory, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. Financial support: Conselho Nacional de Desenvolvimento Tecnolo´gico (CNPq, Brazil), PRONEX-MCT and Fundac- a˜o de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ, Brazil), PAPES4 (FIOCRUZ, Brazil), FIRCA-NIH (TW5531). Corresponding author. Laborato´rio de Imunofarmacologia, Departamento de Fisiologia e Farmacodinaˆmica, IOC, Fundac- a˜o Oswaldo Cruz, Av. Brasil 4365, Manguinhos, Rio de Janeiro, RJ 21045-900, Brazil. Tel.: +55 21 2598 4492; fax: +55 21 2590 9490. E-mail addresses: [email protected] (J.I. Salluh), fbozza@ipec.fiocruz.br (P.T. Bozza). 0952-3278/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.plefa.2007.04.001

Platelet-activating factor (PAF) is a proinflammatory phospholipid produced by a variety of cells including leukocytes and endothelial cells, which participate in inflammatory conditions such as the acute lung injury [1]. The PAF signaling system is a point of connection and augmentation of the thrombotic and inflammatory cascades [2] and PAF is a key mediator of acute lung injury [3]. Previous studies have shown that PAF is increased in BAL fluid of patients with acute respiratory distress syndrome (ARDS) [4] and mediates lung injury

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in a model of endotoxic shock [5] and in a porcine model of pseudomonas-induced lung injury [6]. Nagase et al. [7] observed that mice overexpressing the PAF receptor had increased mortality, whereas those with a deletion of the PAF receptor exhibited only mild lung injury, indicating that PAF is an important mediator of lung injury. Platelet-activating factor acetylhydrolases (PAF-AH) are enzymes that recognize PAF and biologically active PAF-like oxidized lipids as substrates, terminating their signals and thus regulating inflammatory response [8,9]. Its action occurs as PAF is hydrolyzed into a biologically inactive isoform (lyso-PAF). Prior studies of BAL fluid from patients with asthma and lung fibrosis suggested that PAF-AH inactivates PAF released into the airways [10]. Nakos et al. [11] found increased BAL PAF-AH activity as well as PAF levels in ARDS patients when compared to mechanically ventilated controls. Recently, Grissom et al. [12] observed that PAF-AH levels were increased in the BAL fluid of patients with ARDS. Nevertheless the origin of the enzyme recovered from BAL fluid is still unclear and it has been hypothesized that it might result either from leakage of plasma PAFAH into the alveoli or, in contrast, from local production. Clear understanding of this issue is crucial in defining the role of PAF-AH in the pathophysiology of acute lung injury (ALI), as well as the potential role of recombinant PAF-AH in the treatment of ALI [13]. The purpose of this study was to describe the kinetics of PAF-AH activity in the BAL and plasma during the early phase of ALI and to investigate the source of PAFAH present in the BAL fluid.

2. Methods 2.1. Experimental acute lung injury Eight mixed-breed female pigs (Landrace/Large White) with weights ranging from 17 to 20 kg were used in the experiments. After 12 h of food deprivation, the animals were premedicated with midazolam (0.2 mg/Kg; Dormire; Crista´lia, Sa˜o Paulo, Brazil), intubated, and the femoral vein and artery were catheterised. The animals were sedated with a continuous infusion of ketamine (10 mg/kg/h; Ketamina; Crista´lia, Sa˜o Paulo, Brazil), and paralyzed with pancuronium (2 mg/kg/h; Pavulon; Organon, Sa˜o Paulo, Brazil). The animals were initially ventilated (Amadeus ventilator, Hamilton Medical, Switzerland) with assist-control mode, tidal volume (Vt) of 8 ml/kg, respiratory rate of 25, inspired oxygen fraction (FiO2 ¼ 100%) and positive end-expiratory pressure (PEEP) of 5 cm H2O. Arterial pressure, EKG, oxygen saturation and ETCO2 were continuously monitored throughout the experiment. Immediately after acquisition of data (Time 0) oleic acid (0.1 ml/kg initial dose, followed by aliquots of

0.05 ml/Kg) was administered intravenously in six animals and the dose was titrated until the pressure of arterial oxygen (PaO2)/FiO2 ratio was below 200. After lung injury was established a recruitment maneuver was applied with a sustained inflation (30 cm H2O for 40 s) and the PEEP level was titrated to the lowest elastance level. The protocol lasted 6 h and at the end of the experiment animals were sacrificed by means of intravenous injection of 10% KCl. The lungs of two healthy controls were used for immunohistochemical analysis for PAF-AH in lung tissue. Principles of laboratory animal care were followed (NIH publication No.86-23, revised 1985) and the local Ethical Commission for Assessment of Animal Use in Research approved the study protocol. 2.2. Respiratory mechanics and blood gas analysis Elastance, peak airway pressure, esophageal pressure and airway pressures were continuously monitored and recorded as previously described [14]. Blood arterial gases were analyzed (I-STAT system, Abott Laboratories) every 2 h. 2.3. Inflammatory mediator analysis Blood and mini-bronchoalveolar lavage (mini-BAL) were collected every 2 h from the beginning of the procedure. Blood samples (5 ml) obtained from the arterial catheter were collected in tubes containing sodium citrate. The plasma fraction was separated by centrifugation and the samples were frozen and stored at 70 1C for assessment of PAF-AH activity and cytokines analyzes. Mini-BAL was performed by the injection of 10 ml of 0.9% saline into a subsegmental bronchi and lavage obtained by blind sampling technique using the Combicath (Plastimed, USA) as previously described [15]. BAL samples were processed for analysis of leukocytes, total protein, PAF-AH activity and Interleukin (IL)-8 and IL-6. Leukocyte counts were performed in Neubauer chambers after diluting the samples in Turk’s solution (2% acetic acid). Differential leukocyte counts were performed in cytospin smears stained by May– Gru¨nwald–Giemsa. Leukocyte lipid bodies were stained by osmium tetroxide as previously described [16] and enumerated by light microscopy with an  100 objective lens in 50 consecutive leukocytes. Protein content in BAL fluid was quantified by BCA method using bovine serum albumin as standard. 2.4. PAF-AH activity and cytokine measurements PAF-AH activity was measured using a colorimetric method according to the manufacturer’s instructions (Cayman Chemicals, Ann Arbor, MI, USA). IL-8 and

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IL-6 were measured in plasma and BAL samples using enzyme-linked immunoassays (ELISA porcine Duo-set kits, R&D systems). 2.5. Immunohistochemistry for PAF-AH Portions of the inferior lobes were immediately covered with Tissue Tek O.C.T. compound (Milles Scientific Laboratories Ltd., Naperville, IL, USA) and snap frozen in isopentane in a liquid nitrogen bath. These specimens were subsequently stored at 80 1C before processing and cut into 5 mm sections in a cryostat maintained at 20 1C. Tissue sections were deposited onto slides pretreated with poly-L-lysine (Sigma Chemical Co., St Louis, MO, USA), air-dried and fixed for 10 min in acetone at room temperature. Frozen sections were immunostained by indirect peroxidase using two different primary antibodies against PAF-acetylhydrolase. Antibodies used were a polyclonal rabbit anti-human PAF-AH (Cayman Chemical, Ann Arbor, MI, USA) (1:100), and a polyclonal goat anti-human PAF-AH (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) (1:100), which recognize the secreted plasma isozyme and a cytosolic form, respectively (both with cross-reactivity with pig). Briefly, tissue sections were first immersed in 3% hydrogen peroxidase in methanol for 10 min to block endogenous peroxidase activity. After being rinsed in phosphate buffered saline (PBS) containing 0.5% Tween 20 for 10 min, tissue sections were incubated with nonimmune horse serum for 30 min and, subsequently, with the respective primary antibody for 1 h in a humidified chamber at room temperature. Two sections from each sample were incubated with either PBS alone or with rabbit or goat isotypes (concentration-matched) (Dako A/S, Glostrup, Denmark) and served as negative controls, as appropriate. In addition, to control nonspecific binding, competitive inhibition of antiPAF-AH antibodies was performed by preincubating antibodies with human recombinant PAF-AH (generously donated by ICOS Corporation, Bothel, WA, USA) for 1 h at room temperature. This was followed by sequential 15 min incubation with biotinilated link antibody and peroxidase-labelled streptavidin, provided in the LSAB+ kit, HRP (Dako A/S, Glostrup, Denmark). Staining was completed after a 2 min incubation with the substrate chromogen solution containing 3,30 diaminobenzidine. Preparations were lightly counterstained in Harris’s hematoxylin, dehydrated, and mounted in Permount (Fisher Scientific, Pittsburgh, PA). After all sections had been entirely surveyed under optical microscopy, representative pictures were obtained using a computer-assisted image analyser (ImagePro Plus Version 4.1 for Windows, Media Cybernetics, LP, Silver Spring, MD). Data on lung tissue sampling are provided in the ESM.

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2.6. Statistical analysis Kruskal–Wallis and T Mann–Whitney tests were used to compare data from multiple groups. A po0.05 was considered statistically significant. All data are expressed as mean7SE.

3. Results 3.1. Oxygenation and respiratory mechanics in animals with ALI As lung injury was established, we observed a significant decrease in oxygenation in all animals as reflected by the PaO2/FiO2 ratio (Time 0 ¼ 435761 vs. Time 2 ¼ 100721, p ¼ 0.007) (Fig. 1A). The gas exchange decline, as expected, was followed by worsening of pulmonary mechanics as shown by an increase in pulmonary elastance (Time 0 ¼ 45710.2 vs. Time 2 ¼ 124715.5, CmH2O/L p ¼ 0.0022) (Fig. 1B). 3.2. Total neutrophil count and cytokines in plasma and BAL of animals with ALI Oleic acid-induced acute lung injury resulted in a severe pulmonary inflammatory response as demonstrated by increases in total neutrophil count (Time 0 ¼ 0.3170.09  106 vs. Time 6 ¼ 3.670.9  106, p ¼ 0.002) in the BAL fluid (Fig. 1C). Also, a progressive increase in the number of lipid bodies per leukocyte was demonstrated (Time 0 ¼ 4.370.16 vs. Time 6 ¼ 10.470.24, po0.0001) (Fig. 1D). The increase in lipid bodies may be ascribed to the activated state of recruited leukocytes [16,17]. Significant increase in IL-8 concentration in BAL fluid was also observed (Time 0 ¼ 1.1871.06 vs. Time 6 ¼ 4.7671.24, p ¼ 0.02) (Fig. 1E) but could not be demonstrated in plasma samples. Elevation in BAL total protein concentration (Time 0 ¼ 2757119.1 mg/mL vs. Time 6 ¼ 10587 268.3 mg/mL, p ¼ 0.009) was also observed reflecting the changes in vascular permeability of the alveolar/ capillary membrane (Fig. 2A). 3.3. PAF-AH activity in plasma and BAL of animals with ALI Baseline PAF-AH activity in plasma and BAL was extremely low (plasma Time 0 ¼ 0.111970.0092; BAL Time 0 ¼ 070.0007 nmol/ml/min), particularly in the alveolar compartment. We did not observe significant changes in plasma PAF-AH activity during the experiment even as levels in BAL increased (Fig. 2B). PAF-AH activity was normalized by the total protein concentration, in plasma and BAL over time and after normalization remained significantly increased

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Time (h) Fig. 1. Oleic acid challenge induces acute lung injury and inflammation (n ¼ 6): Evolution of impairment in oxygenation (A) and pulmonary elastance (B) There are significant increases in neutrophil count (C), lipid body number (D) and IL-8 levels (E) in bronchoalveolar lavage fluid from animals with ALI throughout the experiment. Results are expressed in mean7 SE.

at the end of the experiment compared to baseline (PAFAH Time 0 ¼ 0.00170.001 nmol/ml/min/g vs 0.0317 0.018 nmol/ml/min/g, p ¼ 0.04) (Fig. 2C). Nevertheless, we still did not detect changes in plasma PAF-AH activity (p ¼ 0.81). There was no significant correlation between PAF-AH activity and protein concentration in BAL fluid at the end of the experiment (r2 ¼ 0.15).

A ratio of changes in PAF-AH activity and BAL protein concentration in the beginning and in the end of experiment was performed. Increases in PAF-AH activity in BAL were greater than increases in BAL protein concentration (PAF-AH Time 6/PAFAH Time 0 ¼ 31.5 vs. protein Time 6/protein Time 0 ¼ 3.77).

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Fig. 2. PAF-AH activity increases in BAL samples after oleic acid-induced ALI (n ¼ 6):Increases in total protein concentration in the BAL fluid were observed (A) while no changes occur in plasma protein concentration. PAF-AH activity in the BAL fluid was increased at the end of the experiment (B) but no changes were detected in plasma activity. When PAF-AH activity was normalized by the protein concentration (C) we did not observe increases in the plasma PAF-AH ratio, (p ¼ 0.8). Results are expressed in mean7 SE.

3.4. Immunohistochemistry for PAF-AH in lungs of animals with ALI and controls Both secreted and cytosolic forms of PAF-AH were predominantly detected in mononuclear cells of all samples studied (Fig. 3). The positively stained mononuclear cells were morphologically compatible with macrophages. Samples from animals with ALI exhibited markedly higher numbers of stained mononuclear cells in the cellular infiltrates and alveolar spaces compared with samples from normal animals without lung injury. In addition, there was a positive staining for the secreted form of PAF-AH in epithelial cells in samples from animals with ALI, but not in those from normal animals. Immunoreactivity to both forms of PAF-AH was completely abolished after preabsorption with an excess of recombinant PAF-AH (Fig. 3F).

4. Discussion In the present work, we describe increased PAF-AH activity in the BAL fluid of animals with ALI during a very early phase of the oleic acid induced damage and inflammation. This experiment was performed in a clinically relevant [18] experimental model of ALI in large sized mechanically ventilated animals. We believe

that the PAF-AH present in BAL fluid is, at least to some extent, produced in the lungs as suggested by positive immunoperoxidase staining for the enzyme both in macrophages and epithelial cells of animals with ALI. The available data on pulmonary PAF-AH activity and source are mainly based on clinical studies of patients with established ARDS [11,12,19]. Although these studies have reported that PAF-AH activity increases in the lungs in response to an acute injurious or inflammatory event, the source of the enzyme is still incompletely defined. Nakos et al. [11] were the first to report that PAF-AH activity in BAL fluid of patients with ARDS was higher than in healthy controls and also described higher PAF-AH activity in the BAL fluid of patients with pneumonia when compared to mechanically ventilated patients without lung injury [20]. Increased PAF-AH activity was also found in the BAL of patients with fat embolism syndrome [21] suggesting that the enzyme is involved in both pulmonary and extrapulmonary ALI. The evidence that increased activity of PAF-AH in the BAL fluid is a result of alveolar flooding with plasma contents [22] was recently reviewed and this hypothesis is supported by the positive correlation between BAL protein and PAF-AH activity of bronchoalveolar lavage demonstrated in a previous study [23]. In the present

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Fig. 3. Cellular PAF-AH is increased in the alveolar compartment after oleic acid-induced ALI: Detection of PAF-Acetylhydrolase in lung sections by immunohistochemistry. Immunostaining with antibody against the cytosolic form of PAF-Ah shows strong signal on mononuclear cells of the cellular infiltrate in the tissue from an animal with ARDS (A), weak staining on few mononuclear cells in a sample from a normal animal (B), and negative staining of the tissue from a normal animal after preincubation of the antibody with recombinant PAF-AH (C). Immunostaining with antibody against the secreted form of PAF-Ah again yielded a strong signal in mononuclear cells in the cellular infiltrate, and epithelial cells in the tissue from an animal with ALI were also positive (D), there was weak staining on few mononuclear cells in lung tissue from normal animals (E), and negative staining of the tissue from an animal with ALI after preincubation of the antibody with recombinant PAF-AH (F) (Original magnification  400). Arrows indicate positive staining of individual cells in lung tissue.

study, we also observed that BAL protein content increased when compared to baseline values. However, we found that the increase in PAF-AH activity in BAL during experimental lung injury was greater than the increase in BAL protein concentration (PAF-AH Time 6/PAF-AH Time 0 ¼ 31.5 vs. protein Time 6/protein Time 0 ¼ 3.77) and that there was no significant

correlation between protein concentration and PAFAH activity in BAL fluid at the end of the experiment (r2 ¼ 0.15). This suggests the presence of an additional mechanism to explain elevation of PAF-AH in the alveolar compartment. Additional evidence to support this view was derived from our observation that the increase in BAL PAF-AH activity occurs only 6 h after

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the initiation of lung injury, while evidence for disruption of the alveolar-capillary membrane integrity already existed within 2 h of oleic acid infusion. This reinforces the possibility of local production of PAF-AH in the alveolar compartment rather than exclusive plasma leakage. Another interesting aspect is the presence of a relatively compartmentalized inflammatory response as demonstrated by elevation of IL-8 that restricted to the alveolar compartment. Changes in IL-8 and IL-6 levels in plasma were negligible (electronic supplementary data). The presence of a compartmentalized response associated with unchanged PAF-AH activity in plasma supports our hypothesis that PAF-AH is produced in the lungs in response to acute lung injury. Currently, there is evidence that intracellular PAF-AH may be released by activated or injured cells in the alveoli during the acute phase of ALI. Earlier in vitro studies demonstrated that monocytes differentiating into macrophages synthesize and secrete the plasma form of PAF-AH [24] and additional observations demonstrate a role for PAF-AH produced by macrophages in controlling local inflammation caused by PAF [2]. Actually, Triggiani and colleagues [10] were the first to describe PAF-AH activity in BAL fluid and showed that BAL fluid PAF-AH is significantly correlated with the number of BAL macrophages and their ability to release PAF-AH both spontaneously and after stimulation with TNF-a. The data provided by our immunohistochemical staining demonstrate that PAF-AH is present in alveolar mononuclear cells and in lung epithelial cells of animals with ALI but not in healthy controls. PAF-AH activity increased in the lungs in the very early phase of ALI, a finding that is in accordance with those of Grissom and colleagues that found mRNA for PAF-AH in the BAL fluid of patients at risk for ARDS. In this situation, activated alveolar macrophages probably synthesize and secrete PAF-AH. Synthesis of PAF-AH by alveolar macrophages may represent a rapid response to lung injury. PAF-AH activity in the lung of ARDS patients may regulate inflammation caused by PAF and related oxidized phospholipids generated during oxygen radical production in the inflammatory response in ARDS. In the present study, PAF-AH was increased in the BAL fluid and was detected by immunoperoxidase staining in macrophages and epithelial cells. Considering these results, we hypothesize that not only macrophages but also epithelial cells, may be a source of production and secretion of PAF-AH. Accordingly, Jehle [25] investigated the role of PAF-AH in a model of hyperoxya-induced lung injury in rats and demonstrated that type II alveolar cells from the lungs were able to secrete PAF-AH. Despite the clarity of the results presented here, we acknowledge there may be some shortcomings. The first

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of them is related to the lack of measurement of PAF production, since PAF levels could provide strong evidence that PAF-AH production occurs as a counter regulatory mechanism to increased PAF production. However, several methods of PAF detection have proven to have variable accuracy and they should be performed immediately at each time point of the experiment. Moreover, PAF has already been shown to be associated with the pathogenesis of ALI in experimental [8] and clinical models [12,21]. Another important aspect is related to the clinical relevance of PAF-AHs now that recent clinical trials have shown no benefit in the administration of recombinant PAF-AH to patients with severe sepsis. In our opinion although randomized clinical trials failed to provide benefit, there were clearly insufficient adequate preclinical studies allowing a profound knowledge of the enzyme’s kinetics. Recent studies show that critically ill patients have wide variation in PAF-AH plasma levels [26]. Actually, PAF-AH activity was not measured in the COMPASS trial [27]. Therefore, if the restoration of PAF-AH levels is beneficial, the investigators were not able to know which patients could have profited from it. In a recent publication Claus et al. [26] observed that endogenous plasma PAF- AH activity increases over time in critically ill patients, especially in those with sepsis and confirmed that plasma PAF-AH activity is a dynamic variable in critically ill patients [26]. Moreover, a recent study from our group demonstrated survival benefit of the administration of recombinant PAF-AH in a clinically relevant experimental model of sepsis when the enzyme’s levels were restored [9] confirming the observation that PAF-AH activity is a dynamic variable in models of critical illness. Such findings reemphasize the importance of studying PAF-AH kinetics in plasma, also in the alveolar compartment, and at tissue level in order to elucidate its role in regulating the inflammatory response.

5. Conclusions In conclusion, we believe the elevation of PAF-AH levels occurring in early ALI may represent a means of counteracting the proinflammatory stimuli provided by the presence of PAF and oxidized phospholipids in the alveolar milieu. The description of temporal variation of PAF-AH kinetics is important, because it could offer valuable information for the adequate timing for therapeutic use of PAF-AH in ALI/ARDS. Although PAF-AH found in the lungs of animals with ALI may be a consequence of alveolar flooding with plasma content, at least part of it seems to be produced and secreted in the lungs by macrophages and epithelial cells. Our findings reemphasize the importance of studying PAF-AH kinetics in plasma and also in the alveolar

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compartment and at tissue level in order to elucidate its role in regulating the inflammatory response. [12]

Acknowledgements We would like to express our gratitude to Alysson R. Carvalho, Edson F. Assis, Joa˜o H. Soares and Fabio O. Ascoli for their substantial assistance in the experimental procedures.

[13]

[14]

Appendix A. Supplementary data [15]

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.plefa. 2007.04.001.

[16]

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