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Abstract |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We have previously described a model of acute lung injury in the mouse in which intravenous administration of lipopolysaccharide (LPS) results in a marked sequestration of neutrophils in the pulmonary microvasculature, although this by itself was not sufficient to induce injury. If the sequestered neutrophils were exposed to zymosan, then a striking increase in pulmonary vascular permeability to albumin was found, suggesting that sequestered neutrophils may produce one or more mediators capable of acting directly on the capillary endothelium. Because activated neutrophils are known to release platelet-activating factor (PAF), we hypothesized that PAF produced locally within the pulmonary capillaries may be the mediator involved. Treatment of mice with the PAF antagonist UK-74,505 prior to administration of zymosan alone or combined LPS and zymosan resulted in a substantial attenuation of lung injury, as measured by the accumulation of extravascular 125I-labeled human serum albumin. UK-74,505 had no effect on neutrophil sequestration as measured by myeloperoxidase activity in whole lung tissue and as assessed by light microscopy. Administration of UK-74,505 after LPS, but before zymosan, was also effective at inhibiting lung injury but again, neutrophil sequestration was unaffected. In contrast, UK-74,505 had no effect on cobra venom factor-induced lung injury and neutrophil sequestration. These data suggest that PAF production is involved in the increases in pulmonary vascular permeability, but not in the sequestration of neutrophils, induced by zymosan alone or by combined LPS and zymosan treatment. Early treatment with PAF antagonists may be beneficial in preventing the development of acute lung injury in humans.
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Introduction |
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Abstract
Introduction
Materials & Methods
Results
Discussion
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Acute lung injury, such as that associated with the adult respiratory distress syndrome (ARDS), is characterized by a sequestration of neutrophils in the pulmonary microcirculation and an increase in microvascular permeability (1, 2) as well as severe alveolar edema formation. Mortality is often the result of impaired gaseous exchange, hyaline membrane formation, and subsequent alveolar fibrosis. Effective therapeutic intervention remains limited, as the manifestation and diagnosis of lung injury may be many hours after the initiation of pathologic events. The causes connected with the development of ARDS can be a consequence of direct pulmonary insult or other nonpulmonary conditions. Around 50% of ARDS cases are due to sepsis and in these patients, platelet-activating factor (PAF) has been detected at elevated levels in the blood (3, 4).
Experimental lung injury in animals can be induced by administration of endotoxin or lipopolysaccharide (LPS), whereby the classic symptoms of lung injury, i.e., pulmonary leukostasis (5) and increased pulmonary vascular permeability (6), are manifested. Increases in PAF-like activity in plasma are also demonstrated in animals treated with endotoxin (10). In these experimental models, PAF receptor antagonists inhibit several features of endotoxin-induced lung injury, including increased pulmonary vascular permeability and pulmonary edema formation (10). However, increased PAF levels are also detected in the bronchoalveolar lavage fluid of trauma patients and nonseptic patients with ARDS (4, 13) and have therefore been implicated as an effector of the associated pathologic changes. Intravenous injection of PAF results in leukopenia with sequestration of neutrophils in the pulmonary vasculature (14), increases in pulmonary vascular permeability (15, 16), and the formation of pulmonary edema (17).
Despite the data obtained in experimental studies, there is limited clinical experience with PAF antagonists in conditions associated with lung injury such as ARDS, particularly that which is not associated with sepsis. In one phase II study, treatment with the PAF antagonist Lexipafant reduced pulmonary dysfunction in acute pancreatitis (18). In another multicenter phase III trial, repeated dosing with the PAF antagonist BN 52021 over several days resulted in a significant reduction in the mortality of patients with severe gram-negative sepsis (19), suggesting that protracted blockade of PAF receptors during the inflammatory cascade may be effective in a subgroup of septic patients; however, lung dysfunction was not measured in this study.
We have previously described a model of acute lung injury in the mouse, wherein intravenous administration of LPS results in a marked sequestration of neutrophils in the pulmonary microvasculature, although this by itself was not sufficient to induce injury, as assessed by the extravascular accumulation of radiolabeled albumin (20). However, if the sequestered neutrophils were exposed to zymosan, a particulate stimulus, then a striking increase in pulmonary vascular permeability to albumin was found. Zymosan itself, like cobra venom factor (CVF) used in other studies (21), induced significant lung injury alone but this was markedly increased by LPS pretreatment. Neutrophils were not identified in the alveolar spaces after LPS and none was found to be in the process of migration across the endothelium. We have therefore suggested that the increased vascular permeability in the lung in response to zymosan was a result of sequestered neutrophils producing one or more mediators capable of acting directly on the capillary endothelium. Because activated neutrophils are known to release PAF (22, 23) and PAF can increase pulmonary vascular permeability in vivo (15), we hypothesized that PAF produced locally within the pulmonary capillaries, in response to stimulation with zymosan, may be the mediator involved.
The potent, selective, and long-lasting PAF antagonist, UK-74,505 (24) was therefore used to investigate the involvement of PAF receptors in mediating all or part of the lung injury induced by a combination of LPS plus zymosan. In addition, the effect of PAF receptor blockade after LPS-induced neutrophil sequestration had occurred was investigated by administering UK-74,505 after LPS and before zymosan treatment. The role of PAF in lung injury induced by zymosan alone was also investigated and compared with lung injury induced by CVF.
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Materials and Methods |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Animals
All studies were conducted on BALB/c female mice (18- 20 g; Harlan, Oxford, UK). Animal experiments were performed in accordance with the Animals (Scientific Procedures) Act 1986, UK.
Skin Assay
To confirm the efficacy and duration of action of UK-74,505 as an antagonist of PAF-induced vascular permeability increases in the mouse, a skin model of local edema
formation was used. Mice were anesthetized with Hypnorm/Diazepam (16 mg/kg, intraperitoneal), shaved of
dorsal skin hair, and the areas for injection were marked out. Iodinated human serum albumin (125I-labeled HSA;
Amersham International, Little Chalfont, UK) was injected intravenously (approximately 0.25 µCi/animal) and
saline or PAF (1 × 10
9 mol/site) was injected intradermally in duplicate in 50-µl volumes. After 30 min, the animals were overdosed with sodium pentobarbitone and
killed by exsanguination. Blood samples were collected
into heparin and the plasma fractions were prepared. The
dorsal skins were removed and skin sites (11-mm diameter)
punched out and counted with plasma samples in a 
counter
(Canberra Packard, Pangbourne, UK). Local edema formation was expressed as microliters of plasma by dividing
the counts per minute in skin sites by the counts per
minute in 1 µl of plasma.
Induction of Experimental Acute Lung Injury
Mice were anesthetized as described above and received an
intravenous injection of saline (7 ml/kg) or LPS (3 mg/kg)
from Escherichia coli 0111:B4 (Sigma, Poole, UK) and left
for 2 h. We have previously demonstrated that this dose of
LPS induces an approximately six-fold increase in neutrophil sequestration in the lung after 2 h (as assessed by examination of electron micrographs) but does not result in
detectable lung injury (20). Zymosan A (10 mg/kg) from
Saccharomyces cerevisiae (Sigma), or saline in control animals, was then injected intravenously and simultaneously
with 125I-labeled HSA (approximately 0.25 µCi/animal). Extravascular 125I-labeled HSA was used as a measure of increased microvascular permeability in lung tissue and its
accumulation was measured after 30 min. At this time
point 131I-labeled HSA (approximately 0.5 µCi/animal),
prepared according to the chloramine-T method (25), was
injected intravenously and allowed to circulate for 5 min;
131I-labeled HSA was used to quantify the intravascular
volume of the lung. The mice were then given sodium pentobarbitone to induce deep anesthesia and were killed by
exsanguination. A blood sample was collected into heparin and the plasma prepared. The lungs were exposed,
removed en bloc, and the activities of 125I-labeled HSA
and 131I-labeled HSA in whole lungs were counted in a counter and compared with that in the plasma. Extravascular albumin accumulated in lung tissue, expressed as microliters of plasma, was calculated as
Extravascular albumin = (total 125I-labeled albumin
volume) 
(intravascular 131I-labeled albumin volume)
where the total 125I-labeled albumin volume is 125I-labeled albumin counts in lung divided by 125I-labeled albumin counts in 1 µl of plasma, and the intravascular 131I-labeled albumin volume is 131I-labeled albumin counts in lung divided by 131I-labeled albumin counts in 1 µl of plasma.
In separate groups of animals, zymosan alone (10 mg/kg) or CVF (50 U/kg; Cordis Laboratories, Miami, FL) was injected intravenously together with 125I-labeled HSA, and increases in pulmonary vascular permeability were assessed after 30 min in the same way as described above. This dose of CVF is similar to that used by Tvedten and coworkers (21) to induce lung injury in mice and was found to be nonlethal in our preliminary studies; 100 U/kg resulted in substantial mortality and the lung injury detected in those that survived for 30 min was no different than that seen with the lower dose of CVF.
Extravascular Lung Water
Additional experiments were conducted to determine the
extravascular lung water (EVLW) in saline or LPS- and
zymosan-treated mice. The calculation of EVLW was
based on a method previously described (26). Briefly, mice
were treated with saline or LPS for 2 h, and then zymosan
for 30 min. Two minutes before the end of the experiment,
a bolus intravenous injection of 125I-labeled HSA was administered to mark the intravascular space. The mice were
then anesthetized and killed by exsanguination as described previously. Exact volume aliquots of whole blood
were weighed. The lungs were removed, blotted dry of excess blood, and weighed. The wet lung was then counted in
a counter along with the blood aliquots. The lungs and
blood samples were then dried in an oven at 80°C, whereupon they were reweighed. Total lung water was calculated as the difference between the wet weight and dry
weight of the lung. EVLW was calculated as the total lung water minus intravascular lung water and expressed as
milligrams of water per milligram dry weight.
Treatment with UK-74,505
The PAF receptor antagonist UK-74,505 (a gift from Dr. J. Parry, Pfizer, Sandwich, UK) was dissolved initially in 0.1 M HCl and further diluted 10-fold in saline. In the skin studies, control animals received a bolus intravenous injection of vehicle (0.01 M HCl), whereas the test group received an intravenous injection of UK-74,505 at a dose of 0.5 mg/ kg (27, 28). Both treatments were given 2 h prior to intradermal injections. This dose was based on considerable experience with the compound in our department (27, 28) and on our preliminary investigations, which revealed this dose to afford maximum inhibition of PAF-induced plasma leakage in skin (data not shown).
In the lung studies, vehicle or UK-74,505 (0.5 mg/kg) was injected intravenously 15 min prior to further intravenous treatment with either zymosan alone or CVF. In combined, sequential LPS and zymosan studies, UK-74,505 was given 15 min before LPS or 15 min before zymosan.
Histology
Following termination, the lungs were exposed and a small catheter was secured into the trachea to permit their inflation with 10% neutral buffered formalin (pH 7.0), until the pleural margins were sharp. The lungs were then removed en bloc and fixed further by immersion in formalin until processing to paraffin wax. Sections (5- to 6-µm) were cut and stained with hematoxylin and eosin for assessment of interstitial edema and sequestration of leukocytes in lung parenchyma and noncapillary microvessels (i.e., those with a diameter larger than capillaries and with a well-demarcated wall).
Tissue Extraction and Measurement of Myeloperoxidase Activity
The extent of neutrophil sequestration was quantified by measuring myeloperoxidase (MPO) activity (29, 30) in whole lung tissue. The lungs of animals that had received LPS plus zymosan, zymosan alone, or CVF were removed, weighed, and frozen in liquid nitrogen. On thawing, the tissue was weighed and homogenized in 0.2% NaCl buffer (pH 4.7) and centrifuged at 260 × g for 10 min. The supernatant was isolated and ultracentrifuged at 100,000 × g for 60 min, and the resultant pellet resuspended in 0.5% hexyldecyltrimethyl-ammonium bromide (HTAB). MPO activity in the pellet was assayed by measuring the change in absorbance (optical density [OD]) at 690 nm using tetramethylbenzidine (1.6 mM) and H2O2 (0.3 mM). Results were expressed as change in OD per gram of tissue.
Statistics
All data were expressed as mean ± SEM. One-way analysis of variance (ANOVA) was used for analysis of the data
groups. The Student-Newman-Keuls correction factor for
multiple comparisons was used as a posttest (InStat 2.01).
Differences were considered significant when P values
were 
0.05.
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Results |
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Introduction
Materials & Methods
Results
Discussion
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Effect of UK-74,505 on PAF-induced Plasma Leakage in Skin
To examine the efficacy and duration of action of UK-74,505 as an inhibitor of PAF-induced vascular permeability increases, a skin model of local edema formation was
used. In mice pretreated for 2 h with the vehicle (0.01 M
HCl, administered intravenously) plasma leakage in skin
sites 30 min after intradermal injection of saline was 1.6 ± 0.2 µl (n = 4) and in sites injected with PAF (1 × 109
mol/site) was 6.3 ± 0.6 µl (n = 4; P < 0.01 compared with
saline). When animals were pretreated for 2 h with UK-74,505 (0.5 mg/kg, administered intravenously), PAF-
induced edema formation was reduced to 1.4 ± 0.2 µl (n = 4; P < 0.01), which was not significantly different from saline values in the same animals (1.3 ± 0.2 µl). These data
show that UK-74,505 is an effective inhibitor of PAF- induced permeability changes in the mouse and that its inhibitory effects last for at least 2 h.
Effect of UK-74,505 on Lung Injury Induced by Combined LPS and Zymosan
In vehicle-treated animals, the accumulation of radiolabeled albumin in extravascular lung tissue in response to subsequent intravenous treatment with saline was 1.1 ± 0.4 µl (Figure 1). Intravenous administration of LPS followed by zymosan 2 h later resulted in a significant increase in extravascular albumin accumulation in lung tissue measured at 2.5 h (P < 0.01); these data are consistent with our earlier findings (20). Furthermore, EVLW in saline-treated controls was not found to be significantly different from that measured in LPS and zymosan-treated mice. EVLW values were as follows: saline = 3.56 ± 0.4 mg water/mg dry weight (n = 4); and in the LPS + zymosan-treated group, 2.59 ± 0.6 mg water/mg dry weight (n = 4).
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The response to combined LPS and zymosan was similar in animals treated with vehicle (Figure 1). However, pretreatment with UK-74,505, 15 min before LPS administration, resulted in 84% inhibition of extravascular albumin accumulation in response to LPS plus zymosan (P < 0.01; Figure 1). Because UK-74,505 is highly selective for PAF (24, 26, 27), these data suggest a critical role for PAF in the full development of lung injury in this mouse model.
To define the stage at which PAF was involved in mediating experimental lung injury, a second series of experiments was conducted in which UK-74,505 (or vehicle) was administered after LPS and 15 min before the zymosan. Under these conditions, albumin accumulation in response to LPS plus zymosan was not significantly altered by vehicle treatment (Figure 2). However, when UK-74,505 was given after LPS but prior to zymosan, the albumin accumulation was reduced by 64% (P < 0.05; Figure 2). Because the antagonist was less effective when administered in this manner, these data suggest that in addition to inducing neutrophil sequestration, LPS also promotes the production of PAF, which may be involved in priming the sequestered neutrophils.
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Effect of UK-74,505 on Lung Injury Induced by Zymosan and CVF
The data presented in Figure 2 indicate that a large proportion of the increase in pulmonary vascular permeability in the lung is the result of PAF production and action in response to zymosan alone. The involvement of PAF as a mediator of vascular permeability induced by zymosan, without LPS pretreatment, was therefore examined. Figure 3 demonstrates that intravenous administration of zymosan (10 mg/kg) induced lung injury over 30 min, as assessed by the extravascular accumulation of albumin, and this was significantly elevated above saline-treated animals. Administration of vehicle did not significantly alter lung injury in response to zymosan alone; however, pretreatment with UK-74,505 significantly decreased zymosan-induced albumin accumulation by 99% (Figure 3); this value was not significantly different when compared with saline-treated controls.
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CVF is reported to induce acute lung injury in the mouse (21). In our hands, intravenous administration of CVF at a dose of 50 U/kg induced lung injury after 30 min that was similar in severity to that seen after zymosan (Figure 4). This response was not significantly altered by pretreatment with vehicle and, in contrast to the findings with zymosan, UK-74,505 pretreatment did not modify CVF-induced lung injury (Figure 4).
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Effect of UK-74,505 on Neutrophil Sequestration
The histologic profile of lungs taken from saline-treated mice was no different in animals that received vehicle. The lungs were of normal appearance, i.e., alveolar walls were clearly demarcated, without apparent leukocyte sequestration or infiltration of the alveolar spaces, and there was no evidence of interstitial or alveolar edema (Figure 5A). In animals treated with zymosan and pretreated with vehicle, there was marked and diffuse sequestration of neutrophils throughout the alveolar-capillary region although there was no migration into the airspaces (Figure 5B). There was some evidence of leukocyte aggregates in noncapillary vessels, but no indication of edema formation in the lung tissue. Pretreatment with UK-74,505 did not appear to modify the appearance of the tissue in response to zymosan treatment and neutrophil sequestration remained marked and diffuse throughout the lung (Figure 5C).
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Pretreatment with UK-74,505 also did not modify the appearance of the lung taken from the LPS- and zymosan-treated group (not shown). The associated diffuse accumulation of cells was unaffected and remained distinct with aggregates of neutrophils still visible. Similarly, the sequestration of the neutrophils in the alveolar region in animals given UK-74,505 after LPS and 15 min before zymosan was not altered and, on inspection, distinct aggregates of neutrophils were still present throughout the lung (not shown). In addition, CVF-induced neutrophil sequestration was not modified in mice pretreated with UK-74,505. Neutrophils were still visible in the alveolar walls and in the lumen of large blood vessels (not shown).
The magnitude of neutrophil sequestration was quantified by assay of MPO activity in lung tissue from mice receiving zymosan alone. This technique has been used extensively as a marker of lung neutrophil burden (30). Zymosan treatment resulted in an approximately 10-fold increase in MPO activity after 30 min (Figure 6) when compared with lung tissue taken from saline-treated controls. Pretreatment with UK-74,505 had no effect on the increased MPO levels, confirming the observations made using light microscopy. LPS plus zymosan treatment resulted in a similar 10-fold elevation of MPO activity when compared with saline-treated controls and this was unaffected by pretreatment with UK-74,505 (data not shown). In addition, UK-74,505 pretreatment had no significant effect on the increase in MPO activity (approximately 10-fold) in lung tissue from animals with CVF (data not shown).
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Discussion |
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Materials & Methods
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Discussion
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Activation of sequestered neutrophils is necessary for the induction of experimental acute lung injury as assessed by measurement of increases in pulmonary vascular permeability, because LPS alone did not induce vascular permeability in mice despite causing an approximately six-fold increase in neutrophil sequestration, as assessed by examination of electron micrographs (21). The increases in vascular permeability were induced in the absence of neutrophil emigration, consistent with the release and action of agents from neutrophils such as oxygen radicals and proteases, or inflammatory mediators including lipids (PAF, leukotriene B4 [LTB4]) and chemokines. Activated neutrophils are known to generate and release PAF in response to phagocytic stimuli (22, 23), and PAF increases microvascular permeability (15). It is possible, therefore, that endogenously generated PAF may induce endothelial cell retraction, although this has not been investigated in the present study. In this way, pulmonary vascular permeability could be increased in response to treatment with zymosan alone and in the combined LPS and zymosan treatment. Pretreatment of mice with LPS prior to zymosan administration significantly increased the pulmonary vascular permeability although extravascular lung water was not altered when compared with saline-treated controls. Assessment of the lung tissue by light microscopy confirmed this finding, because there was no evidence of edema formation in the LPS- and zymosan-treated animals.
The injury response to zymosan and to combined LPS and zymosan treatment was inhibited by the PAF receptor antagonist UK-74,505. Treatment with UK-74,505 did not have a marked effect on the associated intravascular sequestration and aggregation of neutrophils as assessed by either light microscopy or when quantified by assaying MPO activity in lung tissue. These data suggest that administration of zymosan leads to the synthesis and release of PAF, and that this mediator is involved in the early induction of increased permeability in the lung. The inhibition by UK-74,505 of albumin accumulation in extravascular lung tissue when administered after LPS and prior to zymosan is consistent with this suggestion. Because UK-74,505 is a selective PAF antagonist (24, 27, 28), the effects of the compound in the current study can be attributed to PAF antagonism. The lung injury measured is known to be neutrophil dependent, because in mice specifically depleted of circulating neutrophils, treatment with zymosan alone or LPS and zymosan in combination failed to induce the accumulation of extravascular albumin in lung tissue (data not shown). Thus, it is possible that the neutrophil is the cellular source of PAF.
The synthesis and release of PAF were specific to activation with zymosan, because in contrast, pretreatment with UK-74,505 had no detectable effect on CVF-induced vascular permeability changes. This was surprising because it is known that both zymosan and CVF can activate the alternative pathway of the complement cascade and suggests that the involvement of PAF was related to the phagocytosis of zymosan particles. The process of phagocytosis is known to stimulate the secretory processes of the neutrophils (31) and during phagocytosis, PAF formation and release are likely to occur (22, 23). Moreover, zymosan particles can activate their phagocytosis by neutrophils and we have previously found evidence of this process observed in electron micrographs of lung sections, where zymosan particles were identified within neutrophil phagosomes (20). Other studies have shown that pretreatment with catalase and SOD had a protective effect on CVF-induced lung injury (21, 32), although the effects of these agents were not tested in the present study. Oxygen radicals (in particular the hydroxyl radical), probably derived from the activated neutrophil, have therefore been proposed as the initiators of vascular permeability changes in the lung in response to CVF.
When UK-74,505 was administered prior to combined
LPS and zymosan, there was substantial inhibition of the
extravascular accumulation of 125I-labeled HSA in lung tissue. Similarly, blockade of the PAF receptors also led to a
reduction in 125I-labeled HSA accumulation, when UK-74,505 was given after LPS and before zymosan challenge,
although the inhibition of the plasma leakage response
was not as great as that seen when administered before
LPS (64 versus 84% inhibition). These results (shown in Figures 1 and 2) suggest, therefore, that LPS treatment promotes PAF generation. Much of the PAF produced can be
retained by the neutrophil, which has led to the suggestion
that it has a second-messenger or priming role (35). If
PAF were retained by LPS-stimulated neutrophils in mouse
lung, this may explain why LPS alone does not increase
microvascular permeability (20); the predominant role of
the PAF in LPS-stimulated neutrophils in the lung may therefore be priming for enhanced activation/secretion. LPS
also stimulates tumor necrosis factor 
(TNF-) production from macrophages (38) and interleukin-1 (IL-1) production from endothelial cells (39) and these cytokines
may also induce priming of neutrophils (40, 41). Interestingly, because elevated PAF blood levels after endotoxin
administration precede increases in TNF- (5) and because a PAF antagonist inhibits endotoxin-induced TNF- increases (42), it has been suggested that the early generation of PAF plays a crucial role in the subsequent cytokine
production, including that of TNF-. However, such a
pathway may be of limited significance in our model because LPS alone does not result in a vascular leak after
2.5 h (20).
LPS may also stimulate lung microvascular endothelial
cells to generate PAF and a portion of this could remain
associated with the cell surface, where it can be coexpressed with P-selectin on activated endothelium (43, 44).
Thus, on contact of the neutrophil with the endothelium,
surface-associated PAF can activate the neutrophil via the
PAF receptor (45). Subsequent intracellular signaling events
promote the upregulation of CD11/CD18 integrins on the
neutrophil cell surface and increased adhesion to ligands including the intercellular adhesion molecule 1 (ICAM-1).
We believe that in our model, LPS induces the sequestration of neutrophils within the pulmonary microcirculation
at 2 h via 
2 integrins on the neutrophils binding to endothelial ICAM-1. In support of this, monoclonal antibodies
to CD11b or ICAM-1 reduce neutrophil sequestration in
pulmonary capillaries and the lung injury induced by a
combination of LPS and zymosan (20, 46). The PAF antagonist UK-74,505 is the first agent that we have found to
reduce lung injury without an effect on neutrophil sequestration. Neither sequestration nor the aggregation of the
neutrophils in lung tissue was altered by pretreatment with
the PAF antagonist. This is in line with other reports
wherein PAF does not appear to be involved in the adhesion of neutrophils to cytokine-stimulated endothelial cells
(47, 48) and is not involved in endotoxin-induced pulmonary leukostasis (5). The role of P-selectin in the response
to LPS in mouse lung is not known despite the observation
that it is upregulated in lung tissue 1.5 h after LPS (49) and
can be found as early as 5 min (50). It is possible therefore
that LPS upregulates P-selectin, which contributes to neutrophil sequestration and injury in our model, but this remains to be investigated.
We suggest that in addition to inducing the sequestration of neutrophils within the pulmonary microcirculation
via a combination of expression of 2 integrin on the neutrophils (20) and perhaps decreased deformability (51),
LPS also primes the sequestered cells via PAF and cytokines generated by local cells, including the endothelium.
Thereupon, activation by zymosan results in the neutrophils releasing PAF as a mediator that increases vascular permeability to plasma proteins. Although the involvement of PAF in endotoxin-induced pulmonary changes
has been previously demonstrated and antagonists of PAF
receptors have been shown to attenuate increases in pulmonary vascular permeability (10), the intricate role that
PAF production plays in the propagation of lung injury has not been clarified. Furthermore, it will be of value in
future studies to evaluate the role of PAF in lung injury associated with bacteremia. The results of our present study
suggest that early use of a PAF antagonist and prolonged
blockade of PAF receptors, even after the onset of lung injury, would attenuate increases in vascular permeability,
thereby limiting extravasation of proteins in lung tissue in
patients likely to progress to ARDS.
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Footnotes |
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Address correspondence to: J. Miotla, Ph.D., Kennedy Institute of Rheumatology, 1 Aspenlea Road, London W6 8LH, UK.
(Received in original form November 20, 1996 and in revised form June 24, 1997).
Acknowledgments: The authors would like to acknowledge the assistance of S. Lorimer in preparing the H&E sections of lung tissue and A. Rogers for the development of the photographs. UK-74,505 was a kind gift of Dr. J. Parry (Pfizer, UK). This work was supported by the Ministry of Defence (UK) and the National Asthma Campaign (UK).
Abbreviations ARDS, adult respiratory distress syndrome; CVF, cobra venom factor; EVLW, extravascular lung water; HSA, human serum albumin; LPS, lipopolysaccharide; LTB4, leukotriene B4; MPO, myeloperoxidase; PAF, platelet-activating factor.
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References |
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Materials & Methods
Results
Discussion
References
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1. Anderson, R., R. Holliday, A. Driedger, M. Lefcoe, B. Reid, and W. Sibbald. 1979. Documentation of pulmonary capillary permeability in the ARDS accompanying human sepsis. Am. Rev. Respir. Dis. 119: 869-877 [Medline].
2. Tate, R. M., and J. E. Repine. 1983. Neutrophils and the adult respiratory distress syndrome. Am. Rev. Respir. Dis. 128: S52-S59 .
3. Lopez Diez, F., M. Nieto, S. Fernandez-Gallordo, M. Gijon, and M. Sanchez-Crespo. 1989. Occupancy of platelet receptors for platelet-activating factor in patients with septicemia. J. Clin. Invest. 83: 1733-1740 .
4. Sorensen, J., B. Kald, C. Tagesson, and M. Lindahl. 1994. Platelet-activating factor and phospholipase A2 in patients with septic shock and trauma. Intensive Care Med 20: 555-561 [Medline].
5. Chang, S.. 1994. Endotoxin-induced pulmonary leukostasis in the rat: the role of platelet-activating factor and tumour necrosis factor. J. Lab. Clin. Med. 123: 65-72 [Medline].
6. Worthen, G. S., C. Haslett, A. J. Rees, R. S. Gumbay, J. E. Henson, and P. M. Henson. 1987. Neutrophil-mediated pulmonary vascular injury: synergistic effect of trace amounts of lipopolysaccharide and neutrophil stimuli on vascular permeability and neutrophil sequestration in the lung. Am. Rev. Respir. Dis. 136: 19-28 [Medline].
7.
Welsh, C.,
D. Lien,
G. Worthen,
P. Henson, and
J. Weil.
1989.
Endotoxin-pretreated neutrophils increase pulmonary vascular permeability in dogs.
J. Appl. Physiol.
66:
112-119
8. Cardozo, C., J. Edelman, J. Jagirdar, and M. Lesser. 1991. Lipopolysaccharide-induced pulmonary vascular sequestration of polymorphonuclear leukocytes is complement independent. Am. Rev. Respir. Dis. 144: 173-178 [Medline].
9. Kanazawa, M., A. Ishizaka, N. Hasegawa, Y. Suzuki, and T. Yokoyama. 1992. Granulocyte colony stimulating factor does not enhance endotoxin-induced acute lung injury in guinea pigs. Am. Rev. Respir. Dis. 145: 1030-1035 [Medline].
10. Chang, S., C. Feddersen, P. Henson, and N. Voelkel. 1987. Platelet-activating factor mediates hemodynamic changes and lung injury in endotoxin-treated rats. J. Clin. Invest. 79: 1498-1509 .
11. Inarrea, P., J. Gomez-Cambronero, J. Pascual, M. del Carmen, Ponte, L. Hernando, and M. Sanchez-Crespo. 1985. Synthesis of PAF-acether and blood volume changes in gram-negative sepsis. Immunopharmacology 9: 45-52 [Medline].
12. Doebber, T., M. Wu, J. Robbins, B. Choy, M. Chang, and T. Shen. 1985. PAF involvement in endotoxin-induced hypotension in rats: studies with PAF receptor antagonist kadsurenone. Biochem. Biophys. Res. Commun. 127: 799-808 [Medline].
13. Matsumoto, K., F. Taki, Y. Kondoh, H. Taniguchi, and K. Takagi. 1992. Platelet-activating factor in bronchoalveolar lavage fluid of patients with adult respiratory distress syndrome. Clin. Exp. Pharmacol. Physiol. 19: 509-515 [Medline].
14. Angle, M. J., R. N. Pinckaed, H. J. Showell, and L. M. McManus. 1987. Ontogeny of the responsiveness to intravenous platelet-activating factor. Lab. Invest. 57: 321-328 [Medline].
15.
Burhop, K. E.,
J. G. N. Garcia,
W. M. Selig,
S. K. Lo,
H. Van der Lee,
J. E. Kaplan, and
A. B. Malik.
1986.
Platelet-activating factor increases lung
vascular permeability to protein.
J. Appl. Physiol.
61:
2210-2217
16.
Christman, B. W.,
P. L. Lefferts,
G. A. King, and
J. R. Snapper.
1988.
Role of circulating platelets and granulocytes in PAF-induced pulmonary dysfunction in awake sheep.
J. Appl. Physiol.
64:
2033-2041
17. Hamasaki, Y., M. Morajad, T. Saga, H. Tai, and S. Said. 1984. Platelet-activating factor raises airway and vascular pressures and induces edema in lungs perfused with platelet-free solution. Am. Rev. Respir. Dis. 129: 742-746 [Medline].
18. Kingsnorth, A. N., S. W. Galloway, and L. J. Formela. 1995. Randomized, double-blind phase II trial of Lexipafant, a platelet-activating factor antagonist, in human acute pancreatitis. Br. J. Surg 82: 1414-1420 [Medline].
19. Dhainaut, J., A. Tenaillon, Y. Le Tulzo, B. Schlemmer, J. Solet, M. Wolff, L. Holzappel, F. Zeni, D. Dreyfuss, J. Mira, F. de Vathaire, P. Guinot, and the BN5. 2021. Sepsis Study Group. 1994. Platelet-activating factor receptor antagonist BN 52021 in the treatment of severe sepsis: a randomised, double-blind, placebo-controlled, multicenter clinical trial. Crit. Care Med. 22: 1720-1728 [Medline].
20.
Miotla, J. M.,
T. J. Williams,
P. G. Hellewell, and
P. K. Jeffery.
1996.
A role
for the 2 integrin CD11b in mediating experimental lung injury in mice.
Am. J. Respir. Cell Mol. Biol.
14:
363-373
[Abstract].
21. Tvedten, H., G. Till, and P. Ward. 1985. Mediators of lung injury in mice following systemic activation of complement. Am. J. Pathol. 119: 92-100 [Abstract].
22. Sanchez-Crespo, M., F. Alonso, and J. Egido. 1980. Platelet-activating factor in anaphylaxis and phagocytosis. 1. Release from human peripheral polymorphonuclears and monocytes during the stimulation by ionophore A23187 and phagocytosis but not from degranulating basophils. Immunology 40: 645-655 [Medline].
23. Betz, S. J., and P. M. Henson. 1980. Production and release of platelet-activating factor (PAF): dissociation from degranulation and superoxide production in the human neutrophil. J. Immunol. 125: 2756-2763 [Abstract].
24. Parry, M. J., V. A. Alabaster, H. E. Cheeseman, K. Cooper, R. N. deSouza, and R. F. Keir. 1994. Pharmacological profile of UK-74,505, a novel and selective PAF antagonist with potent and prolonged oral activity. J. Lipid Mediators Cell Signalling 10: 251-268 [Medline].
25. Hunter, W. M., and F. C. Greenwood. 1962. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature (London) 194: 495-496 [Medline].
26.
Selinger, S. L.,
R. D. Bland,
R. H. Demling, and
N. C. Staub.
1975.
Distribution of volume of 131I-albumin, 14C-sucrose and 36Cl in sheep lung.
J. Appl. Physiol.
39:
773-779
27. Pons, F., A. G. Rossi, K. E. Norman, T. J. Williams, and S. Nourshargh. 1993. Role of platelet-activating factor (PAF) in platelet accumulation in rabbit skin: effect of the novel long-acting PAF antagonist, UK-74,505. Br. J. Pharmacol. 109: 234-242 [Medline].
28. Sanz, M.-J., V. B. Weg, M. A. Bolanowski, and S. Nourshargh. 1995. IL-1 is a potent inducer of eosinophil accumulation in rat skin: inhibition of responses by a platelet-activating factor antagonist and an anti-human IL-8 antibody. J. Immunol 154: 1364-1373 [Abstract].
29. Williams, F. M., M. Kus, K. Tanda, and T. J. Williams. 1994. Effect of duration of ischaemia on reduction of myocardial infarct size by inhibition of neutrophil accumulation using an anti-CD18 monoclonal antibody. Br. J. Pharmacol 111: 1123-1128 [Medline].
30. Warren, J. S., K. R. Yabroff, D. G. Remick, S. L. Kunkel, S. W. Chesnue, R. G. Kunkel, K. J. Johnson, and P. A. Ward. 1989. Tumor necrosis factor participates in the pathogenesis of acute immune complex alveolitis in the rat. J. Clin. Invest 84: 1873-1880 .
31. Babior, B. M., R. S. Kipnes, and J. T. Curnutte. 1973. The production by leukocytes of superoxide, a potential bactericidal agent. J. Clin. Invest. 52: 741-744 .
32.
Perkowski, S.,
A. Havill,
J. Flynn, and
M. Gee.
1983.
Role of intrapulmonary release of eicosanoids and superoxide anion as mediators of pulmonary dysfunction and endothelial injury in sheep with intermittent complement activation.
Circ. Res.
53:
574-583
33. Till, G. O., K. J. Johnson, R. Kunkel, and P. A. Ward. 1982. Intravascular activation of complement and acute lung injury: dependency on neutrophils and toxic oxygen metabolites. J. Clin. Invest. 69: 1126-1135 .
34. Suzuki, Y., T. Tanigaki, D. Heimer, W. Wang, W. Ross, H. Sussman, and T. Raffin. 1992. Polyethylene glycol-conjugated superoxide dismutase attenuates septic lung injury in guinea pigs. Am. Rev. Respir. Dis. 145: 388-393 [Medline].
35. Worthen, G. S., J. F. Seccombe, K. L. Clay, L. A. Guthrie, and R. B. Johnston. 1988. The priming of neutrophils by lipopolysaccharide for production of intracellular platelet-activating factor. J. Immunol. 140: 3553-3559 [Abstract].
36. Lynch, J. M., and P. M. Henson. 1986. The intracellular retention of newly synthesized platelet-activating factor. J. Immunol. 137: 2653-2661 [Abstract].
37. Tool, A. T. J., A. J. Verhoeven, D. Roos, and L. Koenderman. 1989. Platelet-activating factor (PAF) acts as an intercellular messenger in the changes of cytosolic free Ca2+ in human neutrophils induced by opsonized particles. FEBS Lett. 259: 209-212 [Medline].
38. Nathan, C. F.. 1987. Secretory products of macrophages. J. Clin. Invest. 79: 319-326 .
39.
Stern, D. M.,
I. Bank,
P. P. Naworth,
J. Cassimersis,
W. Kisiel,
J. W. Fenton II,
C. Dinarello,
L. Chess, and
E. A. Jaffe.
1985.
Self-regulation of procoagulant events on the endothelial cell surface.
J. Exp. Med
162:
1223-1235
40. Sullivan, G., H. Carper, J. Sullivan, T. Murata, and G. Mandell. 1989. Both recombinant interleukin-1 (beta) and purified human monocyte interleukin-1 prime human neutrophils for increased oxidative activity and promote neutrophil spreading. J. Leuk. Biol. 45: 389-395 [Abstract].
41. Pabst, M. J. 1994. Priming of neutrophils. In Immunopharmacology of Neutrophils. P. G. Hellewell and T. J. Williams, editors. Academic Press, London. 195-221.
42. Kuipers, B., T. van der Poll, M. Levi, S. J. H. van Deventer, H. ten Cate, Y. Imai, C. E. Hack, and J. W. ten Cate. 1994. Platelet-activating factor antagonist TCV-309 attenuates the induction of the cytokine network in experimental endotoxemia in chimpanzees. J. Immunol. 152: 2438-2446 [Abstract].
43.
Zimmerman, G. A.,
T. M. McIntyre,
M. Mehra, and
S. M. Prescott.
1990.
Endothelial cell-associated platelet-activating factor: a novel mechanism
for signalling intercellular adhesion.
J. Cell Biol.
110:
529-540
44.
Lorant, D. E.,
K. D. Patel,
T. M. McIntyre,
R. P. McEver,
S. M. Prescott, and
G. A. Zimmerman.
1991.
Coexpression of GMP-140 and PAF by endothelium stimulated by histamine or thrombin: a juxtacrine system for
adhesion and activation of neutrophils.
J. Cell Biol.
115:
223-234
45. Honda, Z., M. Nakamura, I. Miki, M. Minami, T. Watanabe, Y. Seyama, H. Okado, H. Toh, K. Ito, T. Miyamoto, and T. Shimizu. 1991. Cloning by functional expression of platelet-activating receptor from guinea pig lung. Nature (London) 349: 342-346 [Medline].
46. Miotla, J., T. Williams, P. Jeffery, and P. Hellewell. 1994. A role for ICAM-1 in mediating neutrophil-induced increased pulmonary vascular permeability in the mouse. Am. Rev. Respir. Dis. 149: A1092 . (Abstr.) .
47.
Kuijpers, T. W.,
B. C. Hakkert,
M. H. L. Hart, and
D. Roos.
1992.
Neutrophil migration across monolayers of cytokine-prestimulated endothelial cells: a role for platelet-activating factor and IL-8.
J. Cell Biol.
117:
565-572
48.
Hill, M.,
I. Bird,
R. Daniels,
M. Elmore, and
M. Finnen.
1994.
Endothelial cell-associated platelet-activating factor primes neutrophils for enhanced
superoxide production and arachidonic acid release during adhesion to but
not transmigration across IL-1-treated endothelial monolayers.
J. Immunol.
153:
3673-3683
[Abstract].
49. Mayadas, T. N., R. C. Johnson, H. Rayburn, R. O. Hynes, and D. D. Wagner. 1993. Leukocyte rolling and extravasation are severely compromised in P-selectin deficient mice. Cell 74: 541-554 [Medline].
50.
Coughlan, A. F.,
H. Hau,
L. C. Dunlop,
M. C. Berndt, and
W. W. Hancock.
1994.
P-selectin and platelet-activating factor mediate initial endotoxin-induced neutropenia.
J. Exp. Med
179:
329-334
51. Erzurum, S. C., G. P. Downey, D. E. Doherty, B. Schwab, E. L. Elson, and G. S. Worthen. 1992. Mechanisms of lipopolysaccharide-induced neutrophil retention: relative contributions of adhesive and cellular mechanical properties. J. Immunol 149: 154-162 [Abstract].
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