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Abstract |
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Abstract
Introduction
Materials & Methods
Results
Discussion
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The present study has investigated the therapeutic potential of a type 4 phosphodiesterase (PDE) inhibitor,
rolipram, in experimental lung injury. Acute lung injury was induced in the mouse by combined treatment
with lipopolysaccharide (LPS; 10 mg/kg, i.v.) and zymosan (3 mg/kg, i.v.), and assessed using extravascular albumin accumulation; neutrophil sequestration in pulmonary capillaries was also measured. The results show that pretreatment with rolipram (5 mg/kg, i.p.) was protective against the induction of lung injury by combined LPS and zymosan; extravascular albumin accumulation was reduced by 89% and
neutrophil sequestration in lung tissue, as assessed by lung myeloperoxidase (MPO) activity was reduced
by 75%. Pretreatment with rolipram also attenuated increases in serum tumor necrosis factor alpha (TNF
)
levels induced by LPS and zymosan treatment, measured after 2.5 h. The role of endogenous TNF in the
induction of lung injury was therefore assessed. Blockade of endogenous TNF by treatment with the soluble receptor p55-IgG fusion protein or an anti-murine TNF monoclonal antibody, TN3.19.12, had no protective effect against LPS and zymosan-induced lung injury. This suggests that there is a disassociation
between TNF production and the induction of injury in this model. Administration of rolipram after LPS
and before zymosan treatment obliterated the increase in pulmonary vascular permeability, but its effect on
sequestration of neutrophils in pulmonary microvessels, as measured by MPO, was less marked. The results of the present study suggest that use of agents such as rolipram that inhibit PDE4 may have a therapeutic role in treatment of acute lung injury, since we have shown that it is effective in attenuation of neutrophil activation even after sequestration. However, its effect appears to be independent of TNF inhibition.
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Introduction |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Acute lung injury or acute respiratory distress syndrome
(ARDS) is associated with a spectrum of medical conditions and is a manifestation of acute vascular disruption.
The sequestration of neutrophils in the pulmonary microcirculation and their activation appears to be a key event
in the development of the lung injury. The sequestered
neutrophils, when activated, are a source of proteases, reactive oxygen species and inflammatory mediators (1).
These products can contribute to pulmonary vascular endothelial cell and alveolar epithelial cell damage and promote increased pulmonary vascular permeability and edema
formation, features of pulmonary dysfunction (1). The ensuing impaired gaseous exchange can be a direct cause of
mortality. The onset of ARDS is often an early symptom
of multiple organ failure associated with sepsis, and this is
associated with elevated blood levels of endotoxin or lipopolysaccharide (LPS). LPS has therefore been implicated as an important inducer of lung injury (2) and experimentally, endotoxin or LPS has been used to induce acute
lung injury in animals (3). LPS has many proinflammatory actions in the lung, including the induction of neutrophil sequestration in pulmonary capillaries, upregulation
of cell adhesion molecules on endothelial cells (8), and
the promotion of cytokine synthesis and release from alveolar macrophages and endothelial cells (11, 12). One of
the principal cytokines induced by LPS is tumor necrosis factor alpha (TNF). This cytokine has been implicated as
a mediator of the pathologic changes encountered in septic shock, because TNF levels are elevated in the plasma,
bronchoalveolar lavage (BAL) fluid, and lung tissue of
septic patients with ARDS (13). Administration of
TNF itself to animals can induce neutrophil sequestration in pulmonary capillaries as well as their activation (16) and can cause pulmonary damage in vivo (19).
It is conceivable, therefore, that attenuation of LPS-
induced lung injury may be achieved by the inhibition of
TNF production (or action) in vivo. Agents which increase intracellular cyclic adenosine 3'5'-monophosphate
(cAMP), such as prostaglandin E1 and phosphodiesterase
(PDE) inhibitors or cAMP analogues, all inhibit TNF production both in vitro (20) and in vivo (23). The intracellular levels of cyclic nucleotides, including cAMP, are regulated by a family of PDE enzymes which degrade them
and render them biologically inactive (24). Inhibition of the
PDE enzymes results in an accumulation of intracellular
cAMP, which leads to a suppression of neutrophil activity,
including chemotaxis, degranulation, and the respiratory
burst (25). The nonspecific PDE inhibitor pentoxifylline
has been shown to be protective in lung injury induced by
endotoxin and by TNF in dogs and guinea pigs (28, 29).
Since the main PDE isoenzyme in cells involved in the inflammatory process is type 4, studies have investigated the
effects of specific inhibitors of PDE4 in LPS-induced organ injury. In one study, the specific PDE4 inhibitor rolipram was reported to attenuate LPS-induced mortality and
gross pulmonary injury in rats, and this was attributed to
suppression of the increases in serum TNF levels (30).
However, the role of endogenous TNF in mediating the
induction of lung injury remains unclear and there seems
to be contention as to whether TNF plays a role in LPS-lung injury.
The aim of the present study, therefore, was to ascertain the effect of PDE4 inhibition on induction of lung injury and to determine possible modes of action. We have
previously described a murine model of acute lung injury,
induced by combined treatment with LPS and zymosan
(31), where we have observed that the induction of injury
is dependent on the activation of sequestered neutrophils. We have therefore investigated the effect of the specific
PDE type 4 inhibitor rolipram on lung injury, as assessed
by increases in pulmonary vascular permeability and neutrophil sequestration, as well as measuring levels of TNF
in serum. In addition, we have assessed the effect of neutralization of endogenous TNF on lung injury by use of a
soluble TNF receptor protein and a specific chimeric antibody against murine TNF.
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Materials and Methods |
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Abstract
Introduction
Materials & Methods
Results
Discussion
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Induction of Experimental Acute Lung Injury
Lung injury was induced in BALB/c female mice (18-20 g, Harlan Olac, Bicester, UK) by injection of LPS from Escherichia coli 0111:B4 (Sigma, Poole, UK) at a dose of 3 mg/kg (i.v.). This dose of LPS has been shown to induce neutrophil sequestration in the lung at 2 h, but does not result in detectable lung injury (31). Control mice received an i.v. injection of saline (7 ml/kg). After 2 h, zymosan A (10 mg/kg) from Saccharomyces cerevisiae (Sigma, UK), or saline in control animals, was then injected i.v. simultaneously with 125I-human serum albumin (HSA) (approximately 250 nCi/animal; Amersham International, Little Chalfont, UK). Extravascular 125I-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-HSA (approximately 500 nCi/animal), prepared according to the chloramine T method (32), was injected i.v. and allowed to circulate for 5 min. 131I-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 fraction was prepared. The lungs were exposed, removed en bloc and the activities of 125I-HSA and 131I-HSA in whole lungs were counted in a gamma counter and compared with those in the plasma. The volume of extravascular albumin accumulated in lung tissue was then calculated by subtracting the tissue 131I-HSA plasma volume from the 125I-HSA plasma volume and was expressed as microliters of plasma equivalents retained in whole lung tissue.
In separate groups of animals, zymosan alone (10 mg/kg) was injected i.v. together with 125I-HSA and extravascular albumin accumulation was assessed after 30 min in the same manner described above.
Treatment with Rolipram
Rolipram was a gift from Dr. J. Fozard, Sandoz, Basel, Switzerland. It was dissolved in ethanol and further diluted in saline to a final concentration of 0.5 mg/ml in no more than 2.5% ethanol. Rolipram was injected i.p., at doses of 1 mg/kg and 5 mg/kg, 30 min prior to further i.v. treatment with combined sequential LPS and zymosan administration. These doses were chosen since they have been shown to be effective in LPS-induced mortality in rats (29) and to reduce neutrophil recruitment into the peritoneal cavity of mice (33). In a separate group of animals, rolipram (5 mg/kg) was administered after LPS treatment and 30 min prior to subsequent zymosan.
Treatment with TNFR-IgG Fusion Protein and mAb TN3.19.12
The soluble human TNF receptor (p55)-IgG fusion protein (TNFR-IgG) was a gift from Drs. Scallon and Ghrayeb, Centocor Inc. (Malvern, PA). Neutralization of endogenous TNF was achieved by i.p. administration of
TNFR-IgG at a dose of 5 mg/kg 6 h before subsequent
LPS and zymosan treatment. Lung injury was measured as
previously described. The dosing regimen has been shown
to be protective against LPS-induced mortality and attains
complete neutralization of circulating TNF in mice (34).
In addition, the anti-TNF monoclonal antibody (mAb)
TN3.-19.12, a gift from Dr. R. Foulkes, Celltech, Slough,
UK, was tested on LPS plus zymosan-induced lung injury.
TN3.19.12 is a murine/hamster chimeric antibody directed
against murine TNF with hamster variable regions and murine heavy and light chain constant regions. Mice were injected i.v. with TN3.19.12 (30 mg/kg) 1 h before treatment with LPS and zymosan, and lung injury was measured as
previously described. This dose of mAb has been previously shown to inhibit LPS-induced mortality in mice by
90% (35).
Histology
In appropriate experiments, after exsanguination, the lungs were exposed and a catheter was secured into the trachea in order to inflate the lungs in situ with 10% neutral buffered formalin (pH 7.0), until the pleural margins were sharp. The lungs were then removed en bloc and further fixed by immersion in formalin until processing to paraffin wax. Sections (5-6 µm) were cut and stained with hematoxylin and eosin for assessment of leukocyte sequestration.
Tissue Extraction and Measurement of Myeloperoxidase Activity
The extent of neutrophil sequestration in whole lung tissue was measured by assaying myeloperoxidase (MPO) activity (36). The lungs of animals that had received LPS plus zymosan with or without rolipram treatment, zymosan alone with or without rolipram treatment, or saline were removed and frozen in liquid nitrogen. Upon thawing, the tissue was 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, whereupon the pellet was resuspended in hexadecyltrimethyl-ammonium bromide. MPO activity in the resuspended pellet was assayed by measuring the change in optical density (O.D.) at 690 nm using tetramethylbenzidine (1.6 mM) and H2O2 (0.3 mM). Results were expressed as change in absorbance (O.D.) per gram of lung tissue.
MPO activity was also assayed in whole lung tissue taken from animals pretreated with either anti-TNF treatment, i.e., TNFR-IgG and TN3.19.12.
Measurement of Serum TNF Levels
Serum samples were prepared from blood taken from control saline-treated mice, LPS and zymosan-treated mice,
and mice pretreated with rolipram prior to combined LPS
and zymosan administration. TNF levels were measured
using a sandwich ELISA kit purchased from Endogen (Bradsure Biologicals, Loughborough, UK). This kit is reported
to detect mouse TNF levels at a concentration of > 10 pg/ml.
Biological activity of TNF in the serum of mice
treated with the anti-TNF treatments was measured by assessing cytotoxicity of the serum on WEHI 164 cells (37).
The WEHI assay was kindly performed by Dr. D. Butler
at the Kennedy Institute of Rheumatology (London, UK)
as previously described (38).
Statistics
All data are 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 post test. Differences were considered significant when probability values were 0.05 or less.
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Results |
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Introduction
Materials & Methods
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Discussion
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Effect of Rolipram Pretreatment on Lung Injury Induced by Combined LPS and Zymosan
Treatment with LPS followed by an i.v. injection of zymosan 2 h later resulted in a significant (P < 0.01) increase in extravascular albumin accumulation in lung tissue when compared with control saline-treated animals (Figure 1a). These data are consistent with our earlier findings (31). The response to LPS and zymosan was not significantly modified by pretreatment with the rolipram vehicle (2.5% ethanol, 10 ml/kg; Figure 1a). However, 30 min pretreatment with rolipram (5 mg/kg) before LPS and zymosan resulted in an 89% inhibition of extravascular albumin accumulation (P < 0.05; Figure 1a). A lower dose of rolipram (1 mg/ kg) had a partial, but nonsignificant, effect on LPS and zymosan-induced injury (7.3 ± 2.8 µl, n = 5) and all further studies were therefore conducted using 5 mg/kg.
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The magnitude of neutrophil sequestration was quantified by assaying MPO activity in lung tissue. Combined LPS and zymosan treatment resulted in MPO activity increasing approximately 8-fold when compared with that in lung tissue taken from saline-treated controls (Figure 1b). Pretreatment with rolipram significantly inhibited this increase by approximately 75% (P < 0.05).
In addition, lung tissue was examined by light microscopy. Treatment with combined LPS and zymosan led to a marked and diffuse sequestration of neutrophils within pulmonary capillaries when compared with saline-treated controls (Figures 2A and 2B). The neutrophils appeared to be intravascular. There was no evidence of intra-alveolar edema formation, which is consistent with our previous finding that the extravascular lung water measured in control saline-treated and LPS and zymosan-treated mice is not significantly different (39). In contrast, there was an apparent decrease in neutrophil recruitment in the lung sections taken from animals treated with rolipram prior to receiving LPS and zymosan (Figure 2C), thus confirming the results obtained for MPO assay.
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Effect of Rolipram Pretreatment on Serum TNF Levels
Levels of TNF were measured in the serum obtained from
saline and LPS and zymosan-treated mice. TNF levels in
saline-treated animals were below the detection limits of
the kit (i.e., < 10 pg/ml). LPS and zymosan treatment resulted in a substantial increase in TNF serum levels to
112 ± 41 pg/ml (n = 3) at 2.5 h. In animals pretreated with
rolipram, the increases in serum TNF induced by subsequent LPS and zymosan were diminished and the levels
were around 25% of those detected in the mice not receiving rolipram (28 ± 8 pg/ml; P = 0.051).
Effect of Blockade of TNF on LPS and
Zymosan-induced Lung Injury
Since lung injury was attenuated by PDE4 inhibition and
this was associated with a reduction in serum TNF levels,
it was hypothesized that neutralization of endogenously
liberated TNF would lead to an attenuation of the induction of injury. We therefore assessed the effect of TNF
neutralization by pretreatment with TNFR-IgG. However,
as can be seen in Figure 3a, 6 h pretreatment with TNFR-IgG (5 mg/kg, i.p.) had no significant effect on LPS and zymosan-induced lung injury, which remained significantly (P < 0.01) elevated above saline-treated controls.
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In addition, the specific anti-murine TNF mAb TN3.-19.12 was used at a dose (30 mg/kg, i.v.) which has been
shown to be efficacious in reducing LPS-induced mortality
in mice. Similar to the findings with TNFR-IgG, pretreatment with TN3.19.12 did not alter extravascular albumin
accumulation in response to LPS and zymosan treatment, which remained significantly (P < 0.01) elevated above saline-treated controls.
Neutrophil sequestration in lung tissue was quantified in animals pretreated with both the mAb TN3.19.12 and the fusion protein TNFR-IgG. MPO activity was increased by approximately 4-fold as a result of LPS and zymosan treatment and was not significantly altered by pretreatment with either TNF blocking treatment (Figure 3b).
The efficiency of anti-TNF treatments in neutralizing
serum TNF activity was assessed in the WEHI assay. Compared with LPS plus zymosan-treated animals with serum
TNF activity of 196 U/ml, TNF activity in the serum of
animals pretreated with either the fusion protein TNFR-IgG or anti-TNF mAb was reduced by approximately 99% and was not significantly different from the levels measured in saline-treated controls (0.6 U/ml).
Effect of Delayed Treatment with Rolipram on Lung Injury Induced by Combined LPS and Zymosan
Since the anti-inflammatory effects of rolipram appeared
to be independent of endogenous TNF, we assessed the
effect of PDE4 inhibition on the capacity of zymosan in
vivo to activate sequestered neutrophils to induce lung injury. Thus, in the next series of experiments, rolipram or
its vehicle was administered after LPS and 30 min before
the zymosan. Under these conditions, the vehicle did not significantly alter albumin accumulation in response to LPS
plus zymosan treatment (Figure 4a). However, when rolipram was given prior to zymosan, the albumin accumulation was abrogated (P < 0.01 compared with LPS and zymosan; Figure 4a) and was not significantly different to
saline-treated controls.
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As previously seen, LPS plus zymosan treatment significantly increased lung MPO activity when compared with saline-treated controls (P < 0.01; Figure 4b). However, in contrast to the effect of rolipram administered 30 min before LPS (see Figure 1b), treatment with rolipram after LPS and 30 min before zymosan resulted in a reduction of MPO levels by only 47% (P < 0.05); this was found to be significantly (P < 0.05) elevated above the MPO levels measured in lung tissue from saline-treated controls.
The histologic profile of lung tissue taken from animals receiving rolipram after LPS and before zymosan confirmed the above data. The extent of neutrophil sequestration appeared to be as marked as in the LPS and zymosan group (see Figure 2D).
Effect of Rolipram Pretreatment on Lung Injury Induced by Zymosan Alone
In separate groups of animals, the effect of rolipram was assessed on lung injury induced by zymosan alone. Pretreatment with the vehicle had no significant effect on zymosan-induced albumin accumulation in whole lung tissue, when compared with lung injury induced by zymosan alone (Figure 5a). However, there was a significant decrease (approximately 86%) in the permeability induced by treatment with zymosan in the animals pretreated with rolipram.
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In addition, quantification of neutrophil sequestration in lung tissue, in response to zymosan treatment by assay of MPO activity, indicated a 5-fold increase when compared with saline-treated controls. This is similar to the magnitude of neutrophil sequestration quantified at the electron microscopic level in pulmonary capillaries after zymosan administration (31). In contrast to its inhibitory effect on vascular permeability, rolipram had a partial (approximately 44%) but nonsignificant inhibitory effect on MPO activity (see Figure 5b).
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Discussion |
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Introduction
Materials & Methods
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Discussion
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Attenuation of experimental lung injury has previously
been shown to be achieved by treatment with nonspecific
inhibitors of PDE. Thus, pentoxifylline leads to a decrease
in neutrophil sequestration and vascular permeability increases induced by LPS treatment (29, 40). However, pentoxifylline is a nonspecific and weak PDE inhibitor and
may also have other actions. More recently, there have been
suggestions that attenuation of TNF production may be
the protective mechanism by which the type 4 PDE inhibitor, rolipram, inhibited LPS-induced lung injury in rats
(30). Attenuation of increases in serum TNF coincided
with a decrease in neutrophil accumulation in lung tissue.
However, the study did not address the role of endogenous TNF in the induction of lung injury. Thus the mechanisms by which PDE4 inhibition can inhibit the induction of lung injury and the role of endogenous TNF in the injury process remain unclear.
It has therefore been the aim of the present study to determine whether a PDE4 inhibitor could also attenuate
lung injury in a mouse model, and to assess whether this is
related to inhibition of TNF production. We report that
pretreatment with the specific PDE4 inhibitor rolipram before LPS administration leads to an attenuation of neutrophil sequestration in pulmonary capillaries as measured by
MPO, an inhibition of lung injury as measured by extravascular albumin accumulation, and a decrease in serum
TNF levels. It has been demonstrated previously that serum TNF after LPS administration is detected within 30 min and has been shown to peak around 90 min in mice
(41). Since production of TNF occurs early after LPS
treatment, TNF, in addition to LPS, may be involved in
the induction of neutrophil sequestration in pulmonary capillaries. TNF is known to upregulate cell adhesion molecule expression on the leukocytes and endothelial cells
(42, 43), and we have previously shown the integrin
CD11b and its ligand ICAM-1 to be involved in the sequestration of neutrophils and the induction of injury in
this model (31, 44).
It was therefore considered important to investigate
the role of endogenous TNF in mediating LPS-induced
lung injury. Neutralization of TNF by TNFR-IgG in the
present study had no significant effect on lung injury. In
addition, use of a specific antimouse TNF antibody also
had no effect either on the LPS and zymosan-induced increases in extravascular accumulation of albumin, or on
the induced sequestration of neutrophils as measured by
the MPO assay. The treatment protocols were exactly as
described in studies in which these reagents inhibited LPS-induced mortality (34, 35) and were effective in neutralizing the biologic activity of serum TNF as assessed by the
WEHI assay. Remick and colleagues (45) reported that
anti-TNF antiserum partially reduced LPS-induced neutrophil sequestration in mice, as measured by lung MPO
activity, but the study did not measure lung injury. In contrast, Gatti and associates (46) reported that anti-TNF
antibodies did not block pulmonary edema or neutrophil
sequestration induced by LPS in mice, but did attenuate
LPS-induced lethality. Thus there appears to be some controversy surrounding the precise role of TNF in the induction of experimental lung injury. Our data on the
mouse are consistent with the study of Pugin and coworkers (47), which reported that the proinflammatory activity
in BAL fluid from ARDS patients was due to interleukin-1
and not TNF.
Since TNF is not involved in the induction of lung injury, the protective effect of PDE4 inhibition must be attributable to other mechanisms. It is possible, for example,
that PDE4 inhibition by treatment with rolipram prior to
LPS can inhibit the upregulation of cell adhesion molecules. The induction of lung injury in this present model
has previously been demonstrated to be dependent on functional expression of CD11b/18 and ICAM-1 (31, 44). Pober
and colleagues (48) have reported that the nonspecific PDE inhibitor isobutyl methylxanthine decreases synthesis and expression of E-selectin and VCAM-1 adhesion
molecules in human umbilical vein epithelial cell cultures
in response to TNF; however, there was no effect on
ICAM-1 expression. In addition, we have found that rolipram is a poor inhibitor of LPS-induced ICAM-1 expression
on human lung microvascular endothelial cells (unpublished observations). In contrast, Derian and associates (49)
found that rolipram inhibited N-formyl-methyl-leucyl-phenylalanine-induced upregulation of the 
2 integrins CD11a
and CD11b on neutrophils. A similar mechanism may contribute to the capacity of rolipram to impair neutrophil
sequestration and the resulting lung injury in the present
model.
Neutrophil sequestration in pulmonary capillaries is also induced when cells are rendered less deformable. In vitro studies have shown that LPS renders neutrophils less deformable, and this is associated with increased assembly of the F-actin filaments (50). Thus, in vivo, the passage of neutrophils through the pulmonary microvasculature and through the lung is likely to be impaired in response to LPS as a result of changes in the neutrophil cytoskeleton. Treatment with rolipram leads to an elevation of intracellular cAMP in circulating leukocytes that may attenuate LPS-induced deformability and prevent the sequestration of neutrophils in pulmonary capillaries. Indeed, cAMP elevating agents render neutrophils more deformable in vitro (51), and this may contribute to the significant reduction of neutrophil sequestration in lung vasculature in rolipram-pretreated mice.
We have previously demonstrated that increases in extravascular albumin accumulation were not detected in animals receiving LPS alone (31). Additional activation of
the neutrophils by zymosan was required to induce lung
injury. The activation process is likely to be independent
of endogenously liberated TNF but associated with systemic complement activation as well as phagocytosis of the
zymosan particles (31). Treatment with rolipram after LPS
and 30 min before zymosan administration led to a complete inhibition of vascular permeability changes, but there
was a less-marked effect on sequestration of neutrophils.
In this model, rolipram was administered after the reported
peak of TNF production and so the effect was likely to
be a direct one, perhaps downregulating neutrophil activation. In addition, rolipram attenuated extravascular albumin accumulation induced by zymosan alone, without
significantly decreasing neutrophil sequestration. In the
present model, therefore, inhibition of PDE4 in vivo directly attenuates neutrophil activation and the ensuing
lung injury. In accordance with reports that PDE4 inhibition reduces neutrophil activation (25), we believe that rolipram attenuates the production of neutrophil-derived
mediators such as superoxide anions, H2O2, and platelet-activating factor (PAF), which are known to increase endothelial permeability (52, 53). Furthermore, we have previously demonstrated that endogenous PAF production is
crucial to the induction of increased vascular permeability
because a PAF antagonist attenuates the measured injury
(39). The finding is an important one because it demonstrates that attenuation of neutrophil activation in the lung
can be achieved regardless of the prevailing conditions.
Finally, it has been suggested that part of the anti- inflammatory effects of rolipram in vivo may be due to its ability to induce the release of endogenous cortisone (54). We have previously shown that a 2-h pretreatment with the steroid dexamethasone effectively inhibited neutrophil sequestration but only partially inhibited increased extravascular albumin accumulation in mouse lung (55). However, dexamethasone was less effective than rolipram at inhibiting extravascular albumin accumulation induced by LPS and zymosan (55) and failed to alter plasma leakage in the lung induced by zymosan alone (unpublished observations). Therefore, although the release of endogenous cortisone may occur after i.p. administration of rolipram, this release is unlikely to account for the marked inhibitory effects of the drug in this mouse lung injury model.
We have demonstrated herein that inhibition of PDE4 has three main anti-inflammatory effects, i.e., attenuation of TNF production, blockade of neutrophil sequestration in pulmonary capillaries, and inhibition of neutrophil activation. The sequestration of neutrophils in pulmonary capillaries occurs rapidly after the onset of sepsis before the development of ARDS. Since it is unlikely that therapeutic intervention could be timed to target the events in this phase, administration of an agent that inhibits activation of neutrophils already sequestered in lung capillaries would therefore be of interest in the clinical condition. In this context, we have shown that a specific PDE4 inhibitor is beneficial in inhibiting induction of lung injury, even after neutrophil sequestration has occurred, and suggest that this class of agents may have utility in ARDS.
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Footnotes |
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Address correspondence to: Paul G. Hellewell, Ph.D., Applied Pharmacology, Imperial College School of Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, UK. E-mail: p.hellewell{at}ic.ac.uk
(Received in original form February 3, 1997 and in revised form June 30, 1997).
Present address for J. Miotla is Kennedy Institute of Rheumatology, 1 Aspenlea Road, London W6 8LH, UK.Present address for M. Teixeira is Departamento de Farmacologia, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
Acknowledgments:
The authors thank Mr. T. Ansari for the preparation of lung
tissue for light microscopy. The WEHI 164 assay was kindly conducted by Dr.
D. Butler at the Kennedy Institute of Rheumatology, London, UK. Rolipram
was a gift from Dr. J. Fozard, Sandoz (Basel, Switzerland). The soluble human
TNF receptor (p55)-IgG fusion protein (TNFR-IgG) was a gift from Drs.
Scallon and Ghrayeb, Centocor, Inc., Malvern, PA. In addition, the anti-TNF
monoclonal antibody TN3.19.12 was donated by Dr. R. Foulkes, Celltech,
Slough, UK. This work has been supported by the Clinical Research Committee, Royal Brompton Hospital; the British Lung Foundation; the National
Asthma Campaign; and Sandoz.
Abbreviations
ARDS, acute respiratory distress syndrome;
cAMP, cyclic adeno-sine 3'5'-monophosphate;
HSA, human serum albumin;
LPS, lipopolysaccharide;
mAb, monoclonal antibodies;
MPO, myeloperoxidase;
PDE, phosphodiesterase;
TNF, tumor necrosis factor alpha;
TNFR, tumor necrosis factor receptor.
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References |
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Materials & Methods
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References
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