1Department of Anesthesiology, and Institutes for 2Surgical Research and 3Anatomy, University of Munich, 81377 Munich, Germany; and 4Institute for Cancer Research, University of Vienna, 1090 Vienna, Austria
Submitted 9 May 2003 ; accepted in final form 11 July 2003
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ABSTRACT |
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acute lung injury; endotoxin; in vivo fluorescence intravital microscopy; leukocyte/endothelial interaction; intercellular adhesion molecule 1
Oxygen radicals, such as and OH·, released by activated leukocytes cause tissue damage by lipid peroxidation, protein modification, and DNA single-strand breaks. In the manifestation of acute lung injury, it has been considered that during DNA repair, the chromatin-bound enzyme poly(ADP-ribose) synthetase (PARS) is activated and catalyzes the transfer of ADP-ribose moieties from NAD to proteins. Because NAD is essential for mitochondrial electron transport, consumption of NAD results in increased ATP demand, hence cellular energy depletion and, ultimately, cell death (5, 33, 37, 43).
In the context of acute lung injury, PARS seems not only to be activated following leukocyte infiltration. It has also been concluded that PARS is involved in the process of pulmonary leukocyte infiltration since, e.g., intratracheal instillation of endotoxin provoked less myeloperoxidase activity in bronchoalveolar lavage of PARS knockout mice compared with wild-type mice (24, 36). However, the underlying microvascular mechanisms of PARS-mediated leukocyte recruitment have still not been clarified in detail.
Retention of leukocytes in the lung is the net result of a dynamic equilibrium between circulating cells and a population of leukocytes that are retarded during their passage through the pulmonary microcirculation. This balance between circulating and resting leukocyte pools may be affected by adhesion molecules, such as ICAM-1, because they mediate a firm attachment of leukocytes on the endothelium of pulmonary venules and, in contrast to systemic microcirculation, of pulmonary arterioles and alveolar capillaries (35). It has been observed that PARS inhibition diminished the pulmonary expression of ICAM-1 induced by zymosan-activated plasma (6). In the lung, in contrast to other organs, there also exist adhesion molecule-independent mechanisms to recruit leukocytes in the pulmonary microcirculation. The reduction of leukocyte deformability due to remodeling of the cytoskeleton following activation may contribute to a mechanical retention of leukocytes within narrow segments of pulmonary microcirculation, especially within alveolar capillaries (12, 27, 41, 42). In addition, changes in microhemodynamics and thus shear forces may influence the exchange between circulating and resting leukocytes and subsequently the tissue concentration of these cells (4, 22, 26, 29). The role of PARS on pulmonary mechanical leukocyte retention and microhemodynamics has not been investigated so far. Correspondingly, the causative relationship between PARS-mediated ICAM-1 expression and leukocyte recruitment in pulmonary microvessels is still not defined.
Therefore, the aim of the present study was to investigate the microvascular mechanism of leukocyte recruitment during systemic inflammation. The leukocyte-endothelial cell interaction was characterized in the lung with regard to the localization in the pulmonary microcirculation and in correlation to mircohemodynamics and, thus shear rates, ICAM-1 expression, and PARS activation. Intravital fluorescence microscopy was used, as this is the only method allowing direct visualization and simultaneous quantification of leukocytes kinetics and microhemodynamic parameters in each segment of the pulmonary microcirculation. In addition, we investigated the development of capillary leakage and edema formation during endotoxemia and/or PARS inhibition.
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METHODS |
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Rabbit preparation. New Zealand White rabbits were anesthetized by application of 50 mg of thiopental sodium iv followed by 50 mg/kg body wt -chloralose. Piritramide (0.5 mg/kg body wt) was given for analgesia and 0.3 mg/kg body wt pancuronium bromide for muscle relaxation. The surgical preparation and experimental setup for intravital microscopy have previously been described in detail (22). Briefly, the animals were tracheotomized, intubated, and pressure-controlled ventilated (FIO2 0.4, inspiratory airway pressure 8 mmHg, expiratory airway pressure 2 mmHg). Catheters were introduced into the carotid and pulmonary artery for continuous measurement of arterial and pulmonary arterial blood pressure. To access the surface of the right lung for intravital microscopy, we partially removed the fourth and fifth ribs, and a transparent window was implanted instead. Subpleural microvessels, in vitro fluorescein isothiocyanate (FITC)-labeled red blood cells (FITC; Sigma, Deisenhofen, Germany), and in vivo Rhodamine 6G-labeled leukocytes (0.3 ml/kg of a 0.2-mmol saline solution of Rhodamine 6G; Merck, Darmstadt, Germany) were sequentially visualized under a fluorescence microscope (Leica, Wetzlar, Germany) during prolonged inspiration periods of 10 s. Video recordings were made from a silicon-intensified video camera (C2400-08; Hamamatsu, Herrsching, Germany) on an S-VHS video recorder (AG-7350; Panasonic, Munich, Germany).
Determination of microhemodynamics, leukocyte kinetics, and edema formation by intravital fluorescence microscopy.Microhemodynamics and leukocyte kinetics in rabbits were analyzed offline from the video recordings using a digital image-processing system (Optimas; Bioscan, Edmonds, WA), as described previously (21). Internal diameters of subpleural arterioles and venules were measured as the closest distance between inner vessel walls. The inner wall of the alveoli was outlined manually on the video monitor by the image analysis system, and the alveolar surface area was determined. Microhemodynamics were described as the harmonic mean of the velocity of at least 30 FITC-labeled erythrocytes passing a predefined vessel cross section or capillary network. Adherent leukocytes were defined as cells not moving for >10 s and expressed per vessel wall surface or per projected alveolar surface, respectively. Edema formation was assessed from the width of alveolar septa determined as the mean closest distance between inner walls of two adjacent alveoli. At least two arterioles, two venules, and five alveolar areas were investigated in each experimental phase.
Determination of ICAM-1 expression, nitrotyrosine formation, and PARS activity by immunohistochemistry. Antigenes in paraffin-embedded lung sections from rabbits were detected with an ABC kit (ABC Kit; Dianova, Hamburg, Germany) according to the manufacturer's instructions. As primary antibodies, anti-ICAM-1 Rb2/3 (murine anti-rabbit monoclonal IgG, generous gift from M. I. Cybulsky) with 1:50 dilution, anti-nitrotyrosine antibody (mouse monoclonal IgG; Upstate Biotechnology, Lake Placid, NY) with 1:50 dilution, and primary anti-poly(ADP-ribose) antibody (mouse monoclonal IgG; Alexis, Lake Placid, NY) with 1:500 dilution were used (6, 18). Lung sections were also incubated with only the primary antibody or with only the secondary antibody to exclude unspecific poly(ADP-ribose) polymerase staining.
Determination of ICAM-1 expression by Western blot analysis. Lung tissues from rabbits and mice were homogenized in 1 ml of lysis buffer (50 mmol Tris, 10 mM EDTA, 1% Triton X, Boehringer Complete, pH 7.4) and centrifuged for 20 min at 15.000 g. Cytoplasmatic protein fractions were separated by the method of Deryckere and Gannon (8). Isolated proteins were preabsorbed with 20 µl of protein A coupled to agarose (Upstate Biotechnology) for 5 min at 4°C. Upon centrifugation at 1.500 rpm, samples were incubated with the indicated antibody for 1 h at 4°C and, after addition of 20 µl of protein A coupled to agarose, incubated overnight at 4°C on a shaker. Subsequently, samples were centrifuged at 2.500 rpm for 5 min at 4°C, solubilized in sample buffer, and boiled for 8 min. Electrophoresis and blotting were performed as published previously (13). As primary antibodies, the anti-rabbit ICAM-1 Rb2/3 [murine anti-rabbit monoclonal IgG, 1:500 dilution, generous gift from M. I. Cybulsky (18)] and anti-mouse ICAM-1 (rat anti-mouse monoclonal IgG, 1:333 dilution; Southern Biotech, Birmingham, AL) were used.
Electron microscopy. For determination of capillary leakage, the extravasation of intravenously infused gold-labeled rabbit serum albumin (RSA) was investigated by electron microscopy. The production of RSA-gold complexes was performed as described previously (19). RSA-gold suspension was infused through a Millipore 0.25-µm filter 10 min before the end of the experiment. After death of the rabbits, the removed lungs were fixed by intratracheal instillation (airway pressure 25 cmH2O) of 1% glutaraldehyde solution (in 0.1 M Na-cacodylate buffer, pH 7.4). Standard electron microscopy technique was used. Tissue was dehydrated and embedded in Epon resin. Sections were cut at 60 nm and mounted on uncoated 200-mesh grids.
Lung wet/dry ratio. To assess pulmonary edema formation, we determined lung tissue wet/dry weight ratio by drying samples at 100°C for 24 h.
Superoxide anion assay. Superoxide anion release from isolated rabbit neutrophils was measured after stimulation with phorbol 12-myristate 13-acetate (PMA) in the combination with the presence or absence of 3-aminobenzamide (3-AB, 2 mg/ml) by the superoxide dismutase-inhibitable reduction of ferricytochrome C as previously described (30).
Myeloperoxidase assay. Measurement of myeloperoxidase activity in lung tissue was performed as described earlier (20).
Experimental protocol. After surgical preparation of the rabbits and confirmation of the exclusion criteria, as mean arterial pressure <65 mmHg, lack of macroscopic visible atelectasis, hemorrhage, or perfusion failure on lung surface, we reinjected autologous FITC-labeled erythrocytes, and a time interval of 30 min was permitted to ensure splenic elimination of rheologically altered, labeled red blood cells (RBC). Rhodamine 6G was injected immediately before the video microscopic pictures. After baseline recordings in the first protocol, the animals were randomly assigned to four groups. The rabbits of the control group received a saline solution [2 ml as bolus and 1 ml/(h · kg · body wt) per infusionem]. To induce a systemic inflammatory response, we applied endotoxin (LPS 0111:B4 from Escherichia coli; Sigma) to the animals of the LPS group [100 µg as bolus and 20 µg/(h · kg · body wt) per infusionem]. The rabbits of the 3-AB group received the PARS inhibitor 3-AB (Sigma) [10 mg as bolus and 5 mg/(h · kg · body wt) per infusionem] (1, 31, 36, 39). The LPS + 3-AB group consisted of rabbits treated with the combination of LPS and 3-AB. 3-AB administration was started 10 min before LPS infusion. After 2 h, the videomicroscopic recordings were repeated. Because the de novo synthesis of adhesion molecules requires several hours, we investigated the leukocyte-endothelial interaction 4-6 h after LPS or LPS + 3-AB infusion in an additional set of experiments. In this second protocol, the surgical preparation started after application of LPS. At the end of the videomicroscopy, the rabbits were killed, and the lungs were removed for immunohistochemistry, Western blot, and electron microscopy.
After randomly assigning them to experimental groups, we gave the mice single doses of 1 mg/kg body wt LPS ip dissolved in PBS. Controls were treated with the pure solvent only. The animals were killed by decapitation at indicated time points posttreatment. The animal tests were performed according to the governmental guidelines for animal care and treatment. Six hours after LPS or solvent administration, mice were killed, and lung tissue removed for Western blot analysis of ICAM-1 protein expression. Lung tissue was immediately shock frozen at liquid nitrogen and stored at -80°C until the analysis.
Statistics. All data are represented as means ± SE. Statistical data analysis was performed using SigmaStat (Jandel, Erkrath, Germany). Comparisons between the groups were tested using ANOVA on Ranks and Dunn's (protocol 1) and Wilcoxon signed-rank test (protocol 2). Repeated measurements were tested by Mann-Whitney rank sum test. Statistical significance was assumed when P < 0.05.
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RESULTS |
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Microhemodynamics. The vessel diameters ranging from 24 to 30 µm did not differ between the groups and did not change over the time either in protocol 1 or in protocol 2 (Figs. 1 and 2). In protocol 1, the RBC velocity remained constant in all groups over 2 h (Fig. 1). In protocol 2, 6 h after the beginning of 3-AB infusion, the LPS-induced decrease of the RBC velocity in arterioles and venules was nearly reversed. In alveolar capillaries, the effect of 3-AB occurred as early as 4 h of treatment with LPS (Fig. 2).
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Leukocyte-endothelial cell interaction. The number of adherent leukocytes in arterioles was significantly increased only in the endotoxin group compared with the control group at 2 h. In postcapillary venules, pretreatment with 3-AB completely prevented the LPS-induced increase of adherent leukocytes. Moreover, there was a significant reduction of adherent leukocytes in the LPS + 3-AB group compared with the LPS group at 6 h in pulmonary arterioles and at 4 and 6 h in venules. Also in alveolar capillary networks, treatment with 3-AB markedly reduced the retention of leukocytes 2, 4, and 6 h after infusion of endotoxin.
Myeloperoxidase activity. Myeloperoxidase activity in lung tissue did not differ between the experimental groups and is summarized in Table 2.
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ICAM-1 expression. Immunohistochemistry of rabbit lung sections from control animals shows constitutive ICAM-1 expression only along the epithelium of pulmonary bronchioles, demonstrating a relatively specific binding of the monoclonal anti-ICAM-1 antibody. After 6 h of LPS infusion, ICAM-1 expression could also be observed along the endothelium of pulmonary vessels and capillaries. Treatment with 3-AB markedly reduced the staining in arterioles/venules and capillaries (Fig. 3). The Western blot analysis of rabbit lung tissue showed a reduction of the ICAM-1 expression in the LPS + 3-AB group compared with the LPS group (Fig. 4). These data were equivalent to the results of the Western blot analysis of the PARS-/- and PARS+/+ mice (Fig. 5).
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Edema formation. At baseline, alveolar septa were on average 19.4 ± 0.9 µm (Fig. 6). Simultaneously with leukocyte sequestration in alveolar capillary networks, the width of alveolar septa increased in the LPS group, indicating the onset of edema formation. This increase was not evident in animals pretreated with 3-AB. Accordingly, gold-labeled albumin detected by electron microscopy was strictly limited to the plasma compartment or to endosomes in polymorphonuclear neutrophils (PMN) and endothelial cells of pulmonary capillaries under control conditions (Fig. 7). In lungs from LPS-treated animals, gold-labeled albumin was detected in the plasma compartment of capillaries, in the interstitium, and in alveoli, indicating protein extravasation. In contrast, PARS inhibition by 3-AB resulted in a nearly complete inhibition of gold-labeled albumin extravasation. These findings indicate reduced edema formation after LPS by PARS inhibition.
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Wet/dry ratio. Two hours after the onset of LPS infusion, the wet/dry ratio (2 h, 3.8 ± 0.1) did not statistically differ from control (2 h, 4.2 ± 0.2). There was also no difference between the wet/dry ratio of the LPS group (2 h, 3.8 ± 0.1 and 6 h, 4.1 ± 0.2) and the LPS + 3-AB group (2 h, 3.9 ± 0.2 and 6 h, 4.1 ± 0.2) at the time points 2 and 6 h.
Nitrotyrosine formation in lung tissue. The interaction of peroxynitrite with proteins results in nitrotyrosine formation, which was assessed in the present study by immunohistochemistry. Anti-nitrotyrosine antibody binding was diffuse in alveolar epithelial cells and in pulmonary vessels of the control group after 6-h infusion of saline solution. Strongly intensified nitrotyrosine formation was observed 6 h after LPS infusion. However, pretreatment with 3-AB effectively abolished the endotoxin-induced production of nitrotyrosine (Fig. 8).
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PARS activity in lung tissue. Poly-ADP-ribosylated proteins were determined by immunohistochemistry as an indicator of PARS activity. In lung sections from control rabbits, only few poly-ADP-ribosylated proteins could be detected (Fig. 9). However, these proteins could be diffusely localized in lung tissue of LPS-treated rabbits (Fig. 9). The additional infusion of 3-AB resulted in a decreased staining of poly-ADP-ribosylated proteins (Fig. 9).
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Superoxide anion production. To evaluate whether 3-AB functions as a radical scavenger, we determined superoxide production by rabbit neutrophils following PMA and/or 3-AB in vitro. As depicted in Fig. 10, incubation with PMA resulted in a significant increase in superoxide anion release, which was not influenced by 3-AB.
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DISCUSSION |
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In vivo microscopy is the only direct approach for investigation of the site and time course of leukocyte hindrance during the passage of these cells through the pulmonary microcirculation and for studying the influence of hemodynamic factors on the interaction of leukocytes with the endothelium. The role of PARS in pulmonary leukocyte recruitment was investigated in our well-established model using a pharmacological inhibitor of PARS instead of PARS knockout mice.
It has been considered that, besides its enzymatic and DNA binding activity, PARS also possesses a coactivator function (16). The competitive inhibition of PARS, compared with genetic deletion of this enzyme, has therefore the advantage that only the ADP-ribosylating ability is blocked but not the DNA-binding activity or the coactivator function. We chose 3-AB, a competitive and common inhibitor of PARS, which is most validated compared with other (even more specific) PARS inhibitors (34).
Pathophysiological sequestration of leukocytes in pulmonary microcirculation was induced in the present study by intravenous infusion of endotoxin. The model of endotoxemia was not aimed to induce severe acute lung injury but to allow for evaluation of mechanisms underlying early leukocyte sequestration in pulmonary microcirculation. The applied dosage [100 µg as bolus and 20 µg/(h · kg · body wt) per infusionem] was chosen since preceding investigations revealed a significant drop of the peripheral leukocyte count without influencing pulmonary macro- and microhemodynamics.
In the present study, a significant increase of adherent leukocytes could be observed in pulmonary arterioles, venules, and alveolar capillaries following LPS infusion compared with control conditions. The localization and dimension of leukocyte adherence following LPS in pulmonary microcirculation are consistent with earlier studies from our group (23) and from Sato et al. (32).
During the inflammatory process, leukocytes may release reactive oxygen and nitrogen species and contribute to peroxynitrite formation. In particular, peroxynitrite alone, with its oxidative potential that is more than ten times higher than that of superoxide anions and nitric oxide, can cause lipid peroxidation and PARS activation. Consistent with this hypothesis, under our experimental conditions the endotoxin-induced pulmonary leukocyte sequestration was associated with elevated staining for nitrotyrosine and ADP-ribose compared with control. It has already been speculated by Liaudet et al. (24) that not only is PARS activated following leukocyte activation, but also that PARS activation may participate in pulmonary PMN recruitment (7, 24, 36), as myeloperoxidase activity seemed to be decreased in lung tissue of PARS knockout mice compared with wild-type mice following intratracheal instillation of LPS. Myeloperoxidase contributes to the antimicrobial system of leukocytes by catalyzing the reaction of hydrogen peroxide mainly with chloride during the neutrophil respiratory burst to form the cytotoxic hypochloric acid. The myeloperoxidase content in neutrophils is in the order of three magnitudes higher than in monocytes. This specificity of myeloperoxidase is used when one assesses the leukocyte count by measuring myeloperoxidase activity. During the respiratory burst, myeloperoxidase is released from leukocytes into tissue. Due to inhibition of myeloperoxidase by other tissue enzymes like catalase, the myeloperoxidase recovery from inflamed tissue is known to be poor (28). This might explain the missing differences of the myeloperoxidase activity between the experimental groups in our model. The role of PARS neither in myeloperoxidase release nor in production of myeloperoxidase inhibitory enzymes has been evaluated so far. Therefore, in the present study, the effect of PARS inhibition on leukocyte recruitment in pulmonary microcirculation was measured by intravital microscopy. The number of adherent leukocytes in pulmonary arterioles and venules in the LPS group nearly doubled that in the LPS + 3-AB group after 2 h. After 6 h, the number of sticking PMN was fourfold greater in the LPS group than in the LPS + 3-AB group. We also demonstrate that PARS might play a role in expression of ICAM-1. This adhesion molecule is constitutively expressed on the endothelium, upregulated upon stimulation of endothelial cells with e.g., endotoxin, and supports the firm attachment of PMN on endothelial cells (2, 9, 25). We found that ICAM-1 expression in lung tissue from rabbits treated with 3-AB and endotoxin was less expressed than in lung tissue from endotoxin-treated rabbits. Expression of ICAM-1 can be initiated by reactive oxygen metabolites. It has been argued that 3-AB functions as a radical scavenger. Thus one can claim that in our model the decrease of ICAM-1 expression in the LPS + 3-AB group compared with the LPS group is not a consequence of PARS inhibition but is the result of 3-AB itself. However, our hypothesis concerning PARS-dependent ICAM-1 expression was confirmed by the difference of ICAM-1 expression between PARS-/- and PARS+/+ mice. In addition, we measured the superoxide anion production of isolated rabbit PMN upon stimulation with PMA in the presence or absence of 3-AB. We did not find any disparities between the results of both groups, indicating that in our model 3-AB seems not to function as a radical scavenger (Fig. 10).
Leukocyte recruitment in pulmonary microcirculation is not only caused by adhesion molecules, such as ICAM-1, but is also influenced by shear forces and by mechanical (adhesion molecule-independent) retention (12, 27, 41, 42). Activated leukocytes reorganize their cytoskeleton by redistribution and elongation of actin microfilaments and hence become stiffer (12). Thus leukocyte passage through narrow segments of pulmonary mirocirculation is impeded, resulting in mechanical retention of leukocytes (12, 27, 41, 42). The influence of PARS on pulmonary microhemodynamics and mechanical retention has not been investigated so far. Therefore, the causative relationship between PARS-mediated ICAM-1 expression and pulmonary PMN recruitment has not been investigated so far. The possibility that PARS regulates the leukocyte recruitment in the analyzed pulmonary arterioles and venules by mechanical retention can be excluded because the diameter of these vessel segments exceeds the diameter of leukocytes by many times. Because no changes in macro- and microhemodynamics occurred, the measured effect of PARS inhibition on leukocyte recruitment presumably results from PARS-regulated expression of adhesion molecules. Because ICAM-1 is upregulated within 2 h, it might be involved in this process (3). After 6 h, the number of sticking PMN was fourfold greater in the LPS than in the LPS + 3-AB group. At this time point the RBC velocity was reduced only in the LPS group, which in conjunction with a further upregulation of adhesion molecule expression may underlie the greater disparities between the groups. In alveolar capillary networks, the predominant site of PMN sequestration in the lung, the number of adherent PMN and the microhemodynamics altered accordingly. Therefore, we suggest that PMN sequestration in alveolar capillary networks seems to be regulated also by adhesion molecules. On the other hand, the influence of PARS inhibition on the reorganization of the PMN cytoskeleton and thus on mechanical PMN alveolar retention remains to be elucidated.
The LPS-induced leukocyte infiltration was associated with a microscopically visible enlargement of alveolar septa, indicating interstitial edema formation. The applied LPS infusion resulted only in a moderate lung injury, because the wet/dry ratio was not increased following LPS compared with control conditions. This widening of alveolar septa resulted from a disturbance of the microvascular barrier, since it was associated with extravasation of gold-labeled albumin into the interstitium and the alveolar space.
In accordance with the beneficial effect of PARS inhibition on pulmonary PMN sequestration after endotoxin, lung edema formation, as shown by decreases of gold-labeled albumin extravasation and width of alveolar septa after LPS + 3-AB, was reduced compared with LPS alone. Our results suggest that PARS activation promotes endotoxin-induced acute lung injury. PARS activation may mediate tissue injury not only by loss of cellular energetics, as proposed recently (38), but also by enhancing the inflammatory process.
To summarize, our data indicate that PARS affects the pulmonary leukocyte-endothelial cell interaction in pulmonary arteries and venules and alveolar capillaries. We first provide evidence that PARS activation mediates the leukocyte sequestration through upregulation of adhesion molecules, as ICAM-1, and not through changes in macro- and microhemodynamics and thus not through shear forces. The inhibitory effect of 3-AB on pulmonary PMN recruitment was associated with a reduction of the development of edema formation during endotoxemia. Therefore, we suppose that pharmacological inhibition of PARS may be a central therapeutic strategy for prevention/treatment of acute lung injury.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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