Phosphodiesterase type 4 inhibitor reduces the retention of polymorphonuclear leukocytes in the lung

Yukio Sato, Shyoko Sato, Tatsuo Yamamoto, Shigemi Ishikawa, Masataka Onizuka, and Yuzuru Sakakibara

Institute of Clinical Medicine, University of Tsukuba, Tsukuba, 305-8575 Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 10-8 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 (10-8 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 10-8 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 (10-8 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In Vitro Studies

F-actin content assay. F-actin content of PMN immediately increased, peaking at 15 s after 10-8 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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Fig. 1.   Effect of rolipram on F-actin content of polymorphonuclear leukocytes (PMN) stimulated with 10-8 M N-formyl-Met-Leu-Phe (FMLP). F-actin content of PMN immediately increased, peaking at 15 s after 10-8 M FMLP stimulation, and remained high for the whole study period. Preincubation of PMN with rolipram reduced this increase of F-actin content in a dose-dependent fashion. Results are expressed as means ± SE of 5 experiments. * P < 0.005 vs. FMLP-stimulated PMN.

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 (10-7 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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Fig. 2.   The passage of PMN through microchannels recorded on a videomicroscope (A-C). FMLP caused plugging of the microchannel with PMN and disturbance of the flow (B). Rolipram reduced the plugging and kept the flow smooth (C).



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Fig. 3.   Effect of rolipram on the transit time of purified PMN stimulated by 10-8 M FMLP. The transit time increased with FMLP stimulation. Preincubation of PMN with rolipram inhibited this increase in a dose-dependent fashion. Results are expressed as means ± SE of 5 experiments. * P < 0.0001 vs. unstimulated PMN; ** P < 0.005 vs. FMLP-stimulated PMN.

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 (10-7-10-5 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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Fig. 4.   Effect of rolipram on the adhesion molecules on PMN stimulated by 10-8 M FMLP. A: L-selectin expression of PMN decreased after FMLP stimulation. Preincubation of PMN with rolipram had no effect on this response. Results are expressed as means ± SE of 5 experiments. * P < 0.005 vs. baseline. B: CD11b expression of PMN increased after FMLP stimulation (P < 0.0001). Preincubation of PMN with rolipram inhibited this increase of CD11b expression in a dose-dependent fashion. Results are expressed as means ± SE of 5 experiments. * P < 0.001 vs. baseline; ** P < 0.05 vs. FMLP-stimulated PMN.

The CD11b expression of PMN increased to 412 ± 53% after FMLP stimulation (P < 0.0001, Fig. 4B). Preincubation of PMN with rolipram (10-7-10-5 M) reduced this increase of CD11b expression in a dose-dependent fashion (10-7 M: 245 ± 26%, P < 0.05; 10-6 M: 191 ± 19%, P < 0.005; 10-5 M: 151 ± 15%, P < 0.005), indicating that rolipram prevented the upregulation of CD11b expression of PMN after FMLP stimulation.

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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Fig. 5.   The dilution curves of 1,1-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled red blood cells (RBC) into aortic root samples of 1 experiment. Cardiac output, calculated using DiI-labeled RBC as a flow indicator, did not change with treatment.

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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Fig. 6.   The dilution curves of DiI-labeled PMN into aortic root samples of 1 experiment. The recovery of all the DiI-labeled PMN from the aortic root samples was calculated and compared with the recovery of DiI-labeled RBC (assumed to be 100%).



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Fig. 7.   The retention of PMN on their first passage through the lung. The retention of PMN increased with FMLP stimulation. Preincubation with rolipram inhibited this increase. Results are expressed as means ± SE of 5 experiments. * P < 0.05 vs. control; ** P < 0.05 vs. FMLP stimulated PMN.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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