Institute of Clinical Medicine, University of Tsukuba, Tsukuba, 305-8575 Japan
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ABSTRACT |
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Phosphodiesterase (PDE) type 4 is the predominant PDE isozyme in polymorphonuclear leukocytes (PMN) and plays a key role in the regulation of PMN activation. The aim of this study was to examine the effect of a PDE type 4 inhibitor, rolipram, on the functional changes and the retention of PMN in the lung. In vitro, F-actin content and L-selectin and CD11b expression of PMN stimulated by N-formyl-Met-Leu-Phe were measured by flow cytometry. PMN deformability was evaluated using silicon microchannels. Rolipram reduced the increase of F-actin and CD11b but did not change the decrease of L-selectin. Rolipram inhibited the increase of the transit time of PMN through the microchannel. We evaluated the retention of PMN in the lung in vivo by infusing labeled blood into the vena cava and examining the recovery into aortic root samples in rabbits. Rolipram inhibited the retention of stimulated PMN in the lung. In conclusion, a PDE type 4 inhibitor, rolipram, reduces the retention of PMN in the lung by reducing deformability change and CD11b upregulation of PMN.
rolipram; neutrophils; actins; adhesion molecules; adenosine 3',5'-cyclic monophosphate
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INTRODUCTION |
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POLYMORPHONUCLEAR LEUKOCYTES (PMN) play important roles in the pathogenesis of acute lung injury, such as acute respiratory distress syndrome (12). One of the initiating events in the development of acute lung injury is the sequestration of activated PMN. Activated PMN sequester in microvessels of the lung because of a decrease in their deformability (13) and an increase of adhesive qualities of PMN and endothelial cells (3).
The decrease in deformability of PMN is mediated by a rapid assembly of filamentous F-actin from soluble G-actin at the cell periphery (10, 16, 24). This results in a prolonged transit time of PMN through the pulmonary capillaries (7), allowing close proximity and adhesion between PMN and endothelial cells. The selectins slow PMN by mediating rolling, and the integrins induce firm adhesion between PMN and endothelial cells (3). The interaction between these adhesion molecules on PMN and their ligands on endothelial cells contributes to prolonged PMN sequestration in lung microvessels (8).
Adenosine 3',5'-cyclic monophosphate (cAMP) is an intracellular messenger that has a suppressant effect on leukocyte activation (22). cAMP is inactivated by phosphodiesterase (PDE), and PDE type 4 is the predominant PDE isozyme in leukocytes (22, 23). PDE type 4 inhibitors increase cAMP level and are reported to inhibit the increase of cytosolic calcium in N-formyl-Met-Leu-Phe (FMLP)-stimulated human PMN (1), which is a pivotal event in PMN activation (21). PDE type 4 inhibitors also suppress endotoxin-induced acute lung injury in mice (11, 18) and in guinea pigs (15). The mechanism of this reduced lung injury induced by PDE type 4 inhibitors is not clear, and we suspect that PDE type 4 inhibition modulates PMN sequestration, a crucial initial step in the pathogenesis of acute lung injury.
Our working hypothesis is that PDE type 4 inhibition reduces PMN sequestration in the lung by changing the functional characteristics of PMN, thereby reducing the lung injury. The aim of this study was to evaluate the effect of the PDE type 4 inhibitor rolipram on PMN activation both in vitro and in vivo. We measured the effect of rolipram on PMN F-actin assembly, deformability, and adhesion molecule expression in vitro using the receptor agonist FMLP. In in vivo experiments we determined the effect of rolipram on the retention of activated PMN in the lungs of rabbits.
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METHODS |
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In Vitro Studies
Cell preparation. Blood samples were collected from healthy volunteers (n = 5) in acid citrate-dextrose. PMN were purified as previously described (19). The PMN purity was >95% with a viability of 98% as assessed by trypan blue exclusion.
F-actin content assay.
Purified PMN were resuspended in Hanks' balanced salt solution
(GIBCO-BRL, Gaithersburg, MD) at a concentration of 2.0 ± 0.1 × 106/ml. PMN were stimulated with
108 M FMLP (Sigma) for 5, 15, 30, 60, 120, and 180 s
at 37°C in the control group. In rolipram-treated groups, PMN were
preincubated with rolipram at different concentrations
(10
5, 10
6, and 10
7 M) for 10 min before FMLP stimulation. The reaction was stopped by fixation with
3% paraformaldehyde for 30 min. F-actin of PMN was stained using
BODIPY FL phallicidin (Molecular Probes, OR) and was measured using a
flow cytometer (FACS Calibur; Becton Dickinson, Franklin Lakes, NJ) as
previously described (19). The change of F-actin content
was expressed as the percent change from the baseline value.
Deformability assay.
PMN deformability was evaluated using a microchannel array flow
analyzer (MC-FAN). The microchannels are made of silicon and resemble
pulmonary capillaries in size (width 6 µm, depth 4.5 µm, length 10 µm). We assessed PMN deformability by measuring the transit
time of 100 µl of purified PMN in Hanks' balanced salt solution
(1.0 ± 0.1 × 106/ml) through the microchannels
with a pressure of 10 cmH2O, and the passage was recorded
on a videomicroscope (17). Three groups were studied:
1) PMN alone; 2) PMN plus FMLP (108
M was added before filtration); 3) PMN plus rolipram plus
FMLP (PMN was incubated with 10
5, 10
6, and
10
7 M rolipram for 10 min and then stimulated with
10
8 M FMLP before filtration).
Adhesion molecules assay.
Whole blood was washed twice in PBS and resuspended in Hanks' balanced
salt solution. The cells were stimulated with 108 M FMLP
for 3 min at 37°C in the control group. In rolipram-treated groups,
PMN were preincubated with rolipram of different concentrations, (10
5, 10
6, and 10
7 M) for 10 min before FMLP stimulation. Changes in the surface expression of CD11b
and L-selectin of PMN were measured by flow cytometry as previously
described (19) using phycoerythrin-conjugated mouse
monoclonal anti-human CD11b antibody (DAKO Laboratories) and
FITC-conjugated CD62L antibody (Pharmingen, San Diego, CA). The change
of CD11b and L-selectin was expressed as the percent changes compared
with the baseline.
In Vivo Study
The effect of rolipram on the retention of FMLP-stimulated PMN in the lung was evaluated by the method described elsewhere in more detail (19).Experimental protocol. Female New Zealand White rabbits (n = 5; weight, 3.0 ± 0.1 kg) were used; all of the experimental procedures were approved by the Experimentation Committee of the University of Tsukuba. All rabbits were anesthetized with ketamine hydrochloride and xylazine. Catheters were placed in the superior vena cava and the aortic root. Heparin (100 U/kg) was administered after surgery.
PMN and red blood cell labeling. PMN and red blood cells (RBC) were simultaneously labeled with Cell Tracker CM-1,1-dioactadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI; Molecular Probe) as described before (19). Blood (9 ml) was collected from the central ear artery of an experimental rabbit into 2 ml of acid citrate-dextrose as anticoagulant. This blood was washed with PMN buffer and centrifuged (1,000 rpm, 8 min). The pellet was resuspended in 40 ml of PMN buffer containing 2 µM CM-DiI and incubated for 5 min at 37° followed by 15 min at room temperature. The labeled cells were washed in PMN buffer, centrifuged, and resuspended in Hanks' balanced salt solution; 93 ± 1% of PMN and 14 ± 2% of RBC were labeled as measured by flow cytometer.
PMN retention on the first passage through the lungs.
Three indicator-dilution runs were carried out in the same animal after
a rapid bolus injection of DiI-labeled RBC and PMN [2.0 ml of labeled
blood was rapidly (1 s) injected into the cranial vena cava]. The
first indicator dilution was done with naïve labeled cells
(control group). The second run was done 30 min after the first run
with an injection of labeled cells that were incubated with FMLP
(108 M) for 1 min ex vivo (FMLP-treated group). The third
run was done 30 min after the second run with an injection of labeled cells that were incubated with rolipram (10
5 M) for 10 min and then with FMLP (10
8 M) for 1 min ex vivo
(rolipram-treated group). The order of indicator-dilution runs
was changed in each rabbit. Blood samples were collected from the
aortic root catheter at 1.0-s intervals in a fraction collector up to
the point of recirculation (9). The DiI-labeled PMN and
RBC in each sample were determined by flow cytometry. The cardiac
output and the retention of PMN in the lung were determined with
DiI-labeled RBC and PMN as indicators (9, 19, 25).
Statistics
Data were analyzed by analysis of variance (ANOVA). The sequential rejective Bonferroni test was used to correct for multiple comparisons (14). F-actin content of PMN and transit time of PMN through microchannel were analyzed by a two-way ANOVA with time as a repeating factor and donor as a grouping factor. A corrected P value < 0.05 was considered significant. All values are expressed as means ± SE. ![]() |
RESULTS |
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In Vitro Studies
F-actin content assay.
F-actin content of PMN immediately increased, peaking at 15 s
after 108 M FMLP stimulation (66 ± 11% increase
from baseline, P < 0.0005), and remained high for the
whole study period (Fig. 1).
Preincubation of PMN with rolipram reduced this increase of F-actin
content in a dose-dependent fashion (with 10
5 M of
rolipram, from 66 ± 11 to 38 ± 7% at 15 s, from
39 ± 8 to 18 ± 6% at 180 s, P < 0.005).
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Deformability assay.
The videomicroscope image shows the plugging of the microchannel with
PMN when FMLP-activated PMN are perfused (Fig.
2B). Rolipram reduced this
plugging (Fig. 2C). The transit time of 100 µl of purified
PMN increased from 42 ± 8 to 341 ± 41 s (Fig. 3, P < 0.0001).
Preincubation of PMN with rolipram inhibited this increase in a
dose-dependent fashion (107 M: 107 ± 26 s,
P < 0.005; 10
6 M: 85 ± 23 s,
P < 0.001; 10
5 M: 72 ± 21 s,
P < 0.0005).
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Adhesion molecules assay of PMN.
The L-selectin expression of PMN decreased by 27 ± 6% after FMLP
stimulation (P < 0.005, Fig.
4A). Preincubation of PMN with rolipram (107-10
5 M) had no effect on this
response, indicating that rolipram did not alter the shedding off of
L-selectin of FMLP-activated PMN.
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In Vivo Studies
RBC first passage through the lung.
The representative dilution curves of DiI-labeled RBC into aortic root
samples from one of five experiments are shown in Fig. 5. Cardiac output was calculated with
DiI-labeled RBC as a flow indicator. The cardiac output did not change
with treatment (control group, 502 ± 34 ml/min; FMLP-treated
group, 478 ± 55 ml/min; rolipram-treated group, 494 ± 40 ml/min). Blood was lost at the rate of 4.7 ± 0.5 ml/run and was
replaced with normal saline; blood pressures remained unchanged during
the experiment.
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PMN first passage through the lung.
Figure 6 shows the representative
dilution curves of DiI-labeled PMN into aortic root samples from
one of five experiments. The recovery of the DiI-labeled PMN from the
aortic root samples was calculated and compared with the recovery of
DiI-labeled RBC (assumed to be 100%). FMLP treatment increased the
retention of PMN on their first passage through the lung from 33.9 ± 9.8 to 67.5 ± 7.4% (P < 0.05). Pretreatment
of PMN with rolipram reduced the retention to 38.1 ± 7.1%
(P < 0.05) (Fig. 7).
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DISCUSSION |
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The results of this study show that a PDE type 4 inhibitor, rolipram, reduced the retention of activated PMN in the lung. This was associated with a reduction of the increase in F-actin content, the decrease in deformability, and the increase in CD11b expression of PMN. These phenotypic and functional characteristics of PMN have all been shown to impact PMN retention in the lung (7, 8). Therefore, selective PDE type 4 inhibition altered PMN functional characteristics that promote the retention in the lung, and we speculate that this could be one of the mechanisms by which PDE type 4 inhibitors such as rolipram attenuate acute lung injury.
The intracellular levels of cyclic nucleotides, including cAMP, are regulated by a family of PDE enzymes that degrade and render them biologically inactive (22). In leukocytes, cAMP is degraded predominantly by PDE type 4 (22, 23). PDE type 4 inhibitors increase cAMP and inhibit PMN activation (22). cAMP prevents the increase of intracellular Ca2+ of PMN (4). The increase of intracellular Ca2+ plays an important role in signal transductions during PMN activation by activating cellular kinases and phosphatases that are important in F-actin assembly and integrin upregulation (4). Therefore, the prevention of Ca2+ influx by rolipram could prevent these responses during PMN activation.
Our study shows that the receptor agonist FMLP rapidly increases the F-actin content of PMN and that rolipram inhibits this increase in a dose-dependent fashion (Fig. 1). This increase in F-actin content with FMLP was associated with a decrease in PMN deformability, measured as an increase in PMN transit time through the microchannels. Rolipram pretreatment inhibited this decrease in PMN deformability after cell activation. The MC-FAN is a device with uniform silicon channels (17), and the average PMN needs to deform to pass through these channels, similar to the case in pulmonary capillary segments (13). The passage of PMN through these channels can be visualized by videomicroscopy (Fig. 2), and the transit times of PMN were compared by this treatment (Fig. 3). We used this PMN transit time as a surrogate marker for PMN deformability and showed that rolipram inhibits the FMLP-induced decrease in PMN deformability. Changes in the cytoskeleton with F-actin assembly at the cell periphery are thought to be responsible for the deformability change of PMN (10, 16, 24). Our results suggest that rolipram inhibits the deformability change of activated PMN by inhibiting F-actin assembly.
Rolipram reduced the increase of CD11b expression of FMLP-stimulated PMN without changing L-selectin shedding (Fig. 4). Rolipram reduces PMN adhesion to the endothelium by inhibiting the expression of CD11b/CD18 of PMN stimulated by FMLP (6). Rolipram also reduces the increase of CD11b and the decrease of L-selectin expression of PMN stimulated by platelet-activating factor (2). Differences in the mechanism of action of different stimuli may explain this different effect of rolipram on L-selectin shedding. Our previous study showed that nitric oxide, which increases guanosine 3',5'-cyclic monophosphate in PMN (20), reduces the increase of CD18 expression of PMN stimulated by zymosan-activated plasma but failed to alter the shedding of L-selectin expression. We suspect that the signaling pathway of L-selectin shedding is different from that of CD11b translocation and that cyclic nucleotides do not influence L-selectin expression on PMN stimulated by FMLP.
The results of our in vivo study show that rolipram inhibited the retention of PMN stimulated by FMLP on their first passage through the lung (Fig. 7). This inhibition observed in vivo is compatible with the results of in vitro studies that show that rolipram reduced the changes of F-actin content, deformability, and CD11b expression of PMN stimulated by FMLP. All of these changes in vitro are important factors in PMN sequestration in the lung. We suspect that these inhibitory effects of rolipram on PMN activation are responsible for the inhibition of PMN retention in the lung. The fluorescent dye used to label the cells forms a stable bond with the cell membrane, and labeled cells are detected by flow cytometry (5). We consider this labeling method to be suitable for investing PMN sequestration in the lung, because it involves minimal cell manipulation in contrast to techniques using radioisotopes.
In summary, the results of this study show that a PDE type 4 inhibitor, rolipram, reduces functional changes in stimulated PMN that promote their sequestration in the lung. Because the sequestration of PMN in pulmonary capillaries is a crucial step in PMN-mediated damage of the lung tissue, this study suggests that PDE4 inhibition could be a feasible early treatment strategy for subjects at risk for acute lung injury.
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ACKNOWLEDGEMENTS |
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The authors thank Dr. Stephan F. van Eeden for reviewing the manuscript and Drs. Yuji Kikuchi and Satoshi Homma for direction in the measurement of PMN transit time through microchannels.
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FOOTNOTES |
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This work was supported by the University of Tsukuba Research Project.
Address for reprint requests and other correspondence: Y. Sato, Univ. of Tsukuba, Institute of Clinical Medicine, 1-1-1 Tennodai, Tsukuba, 305-8575 Japan (E-mail: ysato{at}md.tsukuba.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 1, 2002;10.1152/ajplung.00433.2001
Received 5 November 2001; accepted in final form 18 January 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Anderson, R,
Goolam Mahomed A,
Theron AJ,
Ramafi G,
and
Feldman C.
Effect of rolipram and dibutyryl cyclic AMP on resequestration of cytosolic calcium in FMLP-activated human neutrophils.
Br J Pharmacol
124:
547-555,
1998[Abstract].
2.
Berends, C,
Dijkhuizen B,
de Monchy JG,
Dubois AE,
Gerritsen J,
and
Kauffman HF.
Inhibition of PAF-induced expression of CD11b and shedding of L-selectin on human neutrophils and eosinophils by the type IV selective PDE inhibitor, rolipram.
Eur Respir J
10:
1000-1007,
1997
3.
Carlos, TM,
and
Harlan JM.
Leukocyte-endothelial adhesion molecules.
Blood
84:
2068-2101,
1994
4.
Coffey, RG.
Effects of cyclic nucleotides on granulocytes.
Immunol Ser
57:
301-338,
1992[Medline].
5.
De Clerck, LS,
Bridts CH,
Mertens AM,
Moens MM,
and
Stevens WJ.
Use of fluorescent dyes in the determination of adherence of human leucocytes to endothelial cells and the effect of fluorochromes on cellular function.
J Immunol Methods
172:
115-124,
1994[ISI][Medline].
6.
Derian, CK,
Santulli RJ,
Rao PE,
Solomon HF,
and
Barrett JA.
Inhibition of chemotactic peptide-induced neutrophil adhesion to vascular endothelium by cAMP modulators.
J Immunol
154:
308-317,
1995
7.
Doerschuk, CM.
Neutrophil rheology and transit through capillaries and sinusoids.
Am J Respir Crit Care Med
159:
1693-1695,
1999
8.
Doerschuk, CM.
The role of CD18-mediated adhesion in neutrophil sequestration induced by infusion of activated plasma in rabbits.
Am J Respir Cell Mol Biol
7:
140-148,
1992[ISI][Medline].
9.
Doerschuk, CM,
Allard MF,
Martin BA,
MacKenzie A,
Autor AP,
and
Hogg JC.
Marginated pool of neutrophils in rabbit lungs.
J Appl Physiol
63:
1806-1815,
1987
10.
Frank, RS.
Time-dependent alterations in the deformability of human neutrophils in response to chemotactic activation.
Blood
76:
2606-2612,
1990[Abstract].
11.
Goncalves de Moraes, VL,
Singer M,
Vargaftig BB,
and
Chignard M.
Effects of rolipram on cyclic AMP levels in alveolar macrophages and lipopolysaccharide-induced inflammation in mouse lung.
Br J Pharmacol
123:
631-636,
1998[Abstract].
12.
Hogg, JC.
Felix Fleischner Lecture. The traffic of polymorphonuclear leukocytes through pulmonary microvessels in health and disease.
Am J Roentgenol Radium Ther
163:
769-775,
1994.
13.
Hogg, JC,
and
Doerschuk CM.
Leukocyte traffic in the lung.
Annu Rev Physiol
57:
97-114,
1995[ISI][Medline].
14.
Holland, BS,
and
Copenhaver MD.
An improved sequential rejective Bonferroni test procedure.
Biometrics
42:
417-423,
1987.
15.
Howell, RE,
Jenkins LP,
and
Howell DE.
Inhibition of lipopolysaccharide-induced pulmonary edema by isozyme-selective phosphodiesterase inhibitors in guinea pigs.
J Pharmacol Exp Ther
275:
703-709,
1995[Abstract].
16.
Inano, H,
English D,
and
Doerschuk CM.
Effect of zymosan-activated plasma on the deformability of rabbit polymorphonuclear leukocytes.
J Appl Physiol
73:
1370-1376,
1992
17.
Kikuchi, Y.
Effect of leukocytes and platelets on blood flow through a parallel array of microchannels: micro- and macroflow relation and rheological measures of leukocyte and platelet activities.
Microvasc Res
50:
288-300,
1995[ISI][Medline].
18.
Miotla, JM,
Teixeira MM,
and
Hellewell PG.
Suppression of acute lung injury in mice by an inhibitor of phosphodiesterase type 4.
Am J Respir Cell Mol Biol
18:
411-420,
1998
19.
Sato, Y,
Hogg JC,
English D,
and
van Eeden SF.
Endothelin-1 changes polymorphonuclear leukocytes' deformability and CD11b expression and promotes their retention in the lung.
Am J Respir Cell Mol Biol
23:
404-410,
2000
20.
Sato, Y,
Walley KR,
Klut ME,
English D,
D'yachkova Y,
Hogg JC,
and
van Eeden SF.
Nitric oxide reduces the sequestration of polymorphonuclear leukocytes in lung by changing deformability and CD18 expression.
Am J Respir Crit Care Med
159:
1469-1476,
1999
21.
Sayeed, MM.
Exuberant Ca2+ signaling in neutrophils: a cause for concern.
News Physiol Sci
15:
130-135,
2000
22.
Torphy, TJ.
Phosphodiesterase isozymes: molecular targets for novel antiasthma agents.
Am J Respir Crit Care Med
157:
351-370,
1998
23.
Wang, P,
Wu P,
Ohleth KM,
Egan RW,
and
Billah MM.
Phosphodiesterase 4B2 is the predominant phosphodiesterase species and undergoes differential regulation of gene expression in human monocytes and neutrophils.
Mol Pharmacol
56:
170-174,
1999
24.
Worthen, GS,
Schwab B, III,
Elson EL,
and
Downey GP.
Mechanics of stimulated neutrophils: cell stiffening induces retention in capillaries.
Science
245:
183-186,
1989[ISI][Medline].
25.
Zierler, KL.
Circulation times and the theory of indicator-dilution methods for determining blood flow and volume.
In: Handbook of Physiology. Circulation. Washington, DC: Am. Physiol. Soc, 1962, sect. 2, vol. I, chapt. 18, p. 585-615.