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Second Department of Surgery, Akita University School of Medicine, Akita City, Japan
Correspondence: Hajime Saito, M.D., Ph.D., Second Department of Surgery, Akita University School of Medicine, 1-1-1 Hondo, Akita City 010-8543, Japan. E-mail: hsaito{at}doc.med.akita-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: endothelial cells • neutrophils • Rho
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous studies have shown that calcium-dependent endothelial signaling results in the formation of gaps at the margins between adjacent cells through which neutrophils are able to pass [12 ]. Conversely, recent studies suggest neutrophils bypass endothelial junctions by crossing at tricellular corners, where the margins of three cells converge, and junctions are discontinuous [15 ]. As well, neutrophils can avoid junctions by penetrating and passing through the endothelial cytoplasm [16 ]. Hence, multiple pathways across the endothelium are possible, and the endothelial-dependent mechanisms regulating neutrophil migration are not fully understood.
Conventional, nonmuscle myosin (myosin II) is involved in such basic cellular processes as stress fiber formation and maintenance of the cortical actin layer [17 ] and is stimulated by phosphorylation of myosin light chain (MLC) on serine 19 by calcium/calmodulin-dependent MLC kinase (MLCK), which facilitates the interaction of myosin with actin, leading to the actomyosin-contractile response [18 ]. The phosphorylation state of MLC also appears to be regulated by Rho, a small G-protein that triggers a multiple signaling pathway [19 ]. Rho is a monomeric, 21-kDa GTPase that exists in at least three isoforms in mammals (Rho A, Rho B, and Rho C). Together with Rac, Cdc42, Rho D, Rho E, Rho G, and TC10, they form the Rho GTPase family of proteins, which consists of at least 14 known members having distinct but related functions [20 , 21 ]. For example, Cdc42 is involved in the regulation of the actin-based cytoskeletal structure and the formation of peripheral filopodial extension [22 ]; Rac regulates formation of lamellipodia and membrane ruffling [23 ]; and Rho increases focal adhesion, formation of actin stress fibers [24 ], and cell contraction after thrombin stimulation [25 ]. It is thus well-established that in mammalian cells, Rho/Rac/Cdc42 proteins primarily regulate cytoskeletal reorganization in response to extracellular signals.
Rho also stimulates Rho kinase, which phosphorylates the myosin-binding subunit (MBS) and inactivates myosin phosphatase [26 ], an enzyme that otherwise binds to phosphorylated MLC via MBS and dephosphorylates it. In addition, Rho kinase, like MLCK, phosphorylates MLC on serine 19 [27 ], suggesting that it stimulates myosin-contractile activity. Rho kinase may thus play a key role in regulating the phosphorylation status of MLC. With these observations as background, we have been examining the mechanism by which endothelial Rho mediates the leukotriene B4 (LTB4)-induced migration of neutrophils across a human umbilical vein endothelial cell (HUVEC) monolayer, especially via the actomyosin system. We hypothesize that such migration is regulated not only by calcium/calmodulin-dependent MLCK but also via a Rho-Rho kinase pathway.
Clostridium botulinum exoenzyme C3 transferase is a bacterial toxin that specifically inhibits members of the Rho subfamily by adenosine 5'-diphosphate (ADP) ribosylating an asparagine (Asn-41) at the GTP-binding site [28 29 30 ]. Y-27632 is a specific Rho kinase inhibitor that prevents formation of filamentous actin (F-actin) stress fibers [31 , 32 ]. The aim of the present study was to use these two inhibitors, as well as ML-7, an MLCK inhibitor, to assess the role of endothelial Rho and Rho kinase in LTB4-induced transendothelial neutrophil migration in vitro. The results showed that neutrophil migration was prevented by each of the inhibitors, which antagonized endothelial actin polymerization, myosin II rearrangement, and MLC phosphorylation. Rho and Rho kinase thus appear to be key regulators of transendothelial neutrophil migration.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture
HUVECs were purchased from Dainippon Pharmaceutical Company (Osaka, Japan). The cells were plated in type I collagen (Sigma Chemical Co.)-coated 25 cm2 plastic tissue-culture flasks and maintained in M199 (Life Technologies, Grand Island, NY) supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin G, 100 mg/ml streptomycin, and 2 mg/ml amphotericin B at 37°C under a humidified atmosphere of 5% CO2, 95% air. Confluent HUVECs were detached with Hanks’ buffered saline solution (HBSS) containing 0.05% trypsin/5 mM EDTA (Life Technologies) and seeded onto 12-mm or 30-mm transwell culture chambers containing polycarbonate membrane inserts (pore size, 0.4 mm; Millicell PCF®, Millipore Corporation, Bedford, MA). Before seeding, the culture chamber was coated with glutaraldehyde-cross-linked gelatin using the procedure of Burns et al. [15
]. Briefly, the polycarbonate membrane of the cell-culture chamber was incubated in phosphate-buffered saline (PBS) containing 1% gelatin (Sigma Chemical Co.) for 30 min at 37°C and then fixed for 25 min in PBS containing 0.5% glutaraldehyde. Thereafter, at room temperature, the cells were washed three times in PBS, incubated with 0.1 M glycine, washed again three times in PBS, and stored for up to 1 week in medium. The medium bathing the culture chamber was replaced twice weekly. HUVECs generally reached confluence within 3 days and were used for experiments beginning 7 days after plating. Confluence was confirmed by measuring electrical resistance [12
, 34
] with an Epithelial Voltohmmeter® (World Precision Instruments, Sarasota, FL). Our finding that the electrical resistance of the confluent HUVEC monolayers was 9.0 ± 2.1 
/cm2 was consistent with earlier findings [34
].
Neutrophil isolation
Human neutrophils were isolated from heparinized (10 U/ml), normal venous blood by dextran sedimentation followed by Lymphoprep gradient centrifugation and hypotonic lysis of erythrocytes, as previously described [35
]. Isolated neutrophils were washed and resuspended in polymorphonuclear neutrophil (PMN) buffer (PBS containing 5 mM glucose, pH 7.4) at a concentration of 107 PMN/ml and were kept at room temperature until used in the experiment.
Transendothelial migration assay
We showed that LTB4 acts a chemoattractant, inducing transendothelial neutrophil migration in a time- and concentration-dependent manner, and that maximal effects are obtained 30 min after addition of 10-7 M LTB4 [13
]. In the present study, unless otherwise indicated, purified 107 PMN/ml were added to the upper compartment of the transwell chamber on which HUVECs had been seeded and bathed in M199 medium supplemented with 10% FCS [PMN-to-endothelial cell (EC) ratio of 10:1; ref. 12
]. LTB4 (10-7 M) was added to the lower compartment, and the chamber was incubated for 30 min in a humidified incubator (37°C, 5% CO2, 95% air). Thereafter, the chamber was washed three times with cold PBS to remove nonadherent cells and was fixed with 2.5% glutaraldehyde in PBS (pH 7.3) overnight. The gelatin was then carefully removed and embedded in paraffin, and 4-mm-thick cross-sections were prepared and stained with hematoxylin/eosin. The number of neutrophils beneath the endothelial monolayer (migrated neutrophils) and those on the endothelial monolayer (adherent neutrophils) were counted under a microscope and expressed as the number of neutrophils per 5 high-power fields (hpf).
F-actin and myosin II immunofluorescence
HUVECs were cultured on a 1% gelatin-coated, 12-mm-diameter polycarbonate membrane chamber (Millipore) bathed in medium. After the HUVECs reached confluence, isolated neutrophils were added to the upper compartment, 10-7 M LTB4 was added to the lower compartment, and the chamber was incubated in the humidified incubator (37°C, 5% CO2, 95% air) for 5–60 min and fixed in 3% buffered formalin (pH 7.0).
To assess the F-actin formation, HUVECs were stained with rhodamine-phalloidin. Briefly, each membrane was removed from its chamber and incubated for 120 min at room temperature in PBS containing 10 U/ml rhodamine-phalloidin, 0.1% Triton X-100, and 1% bovine serum albumin (BSA). The membrane was then washed with PBS and covered with 90% glycerol/10% PBS containing 0.1 M n-propylgallate and a coverslip.
For myosin II staining, the membrane was fixed for 1 min in 1% buffered formaldehyde (pH 6.5) and then for 60 min in 2% formaldehyde in PBS containing 0.2% Triton X-100 and 0.5% DOC (sodium deoxycholate). After washing three times with PBS, the membrane was incubated for 2 min with 10 mM sodium borohydrate and then for 60 min in PBS containing 1% BSA. Thereafter, the membrane was immunolabeled for 120 min at room temperature with antimyosin II serum diluted in PBS-1% BSA (final antibody concentration, 0.75 mg/ml). After rinsing with PBS-0.1% BSA, the membrane was immunolabeled for 60 min at room temperature with rhodamine-conjugated anti-rabbit IgG polyclonal antibody. The membrane was then washed again with PBS and covered with 90% glycerol/10% PBS containing 0.1 M n-propylgallate and a coverslip. Prepared membranes were observed using a confocal laser-scanning microscope system (LSM 410, Zeiss, Germany) incorporating an Axiovert 135 fluorescence microscope (Zeiss).
HUVEC MLC phosphorylation
To analyze myosin phosphorylation, myosin was immunoprecipitated as described by Wysolmerski and Lagunoff [36
, 37
] with minor modifications and was evaluated by Western blotting using anti-P Ser and anti-P Thr mAb as probes. After carrying out neutrophil migration assays, the HUVEC monolayer was washed three times with PBS, after which the cells were harvested by exposing them to 0.25% trypsin/5 mM EDTA three times for 10 s each and then rinsed three times with ice-cold HBSS. The collected endothelial cells and neutrophils were isolated on Histopaque density gradients (Sigma Chemical Co.) using the manufacturer’s protocols with slight modification. First, 3 ml histopaque-1119 was added to a 15 ml polypropylene centrifuge tube, after which 3 ml histopaque-1077 was carefully added onto the histopaque-1119. Collected samples (6 ml) were then carefully added to the upper gradient and centrifuge at 700 g for 30 min in a cold room. After centrifugation, the endothelial cell layer was above the histopaque-1077, and the neutrophils were at the interface of the histopaque-1077 and histopaque-1119. The endothelial cells were then transferred to a tube containing 5 ml HBSS and were washed by centrifugation at 200 g for 10 min. The purity of isolated endothelial cells was over 95%.
The isolated endothelial cells were lysed by incubation for 30 min on ice in 400 µl lysis buffer (25 mM Tris-HCl, pH 7.9, 250 mM NaCl, 100 mM Na2P2O7, 75 mM NaF, 0.5% sodium deoxycolate, 1% Nonidet P-40, 5 mM EGTA, 5 mM EDTA, 0.2 µM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin), after which the soluble cell extract was centrifuged at 132,000 g for 10 min, and the supernatant was collected. The pellet was extracted again by incubation for 20 min in 200 µl lysis buffer, this time containing 600 mM NaCl. The insoluble materials were again sedimented at 132,000 g, and the extracts were diluted with an equal volume of lysis buffer without NaCl and then combined with the initial sample. To avoid nonspecific binding, the sample was preincubated for 30 min at 4°C with 100 µl 20% skim milk (pH 7.4) and 20 µl 50% protein A/G sepharose, after which the sample was centrifuged for 10 min at 15,000 g, and the supernatant was collected.
The collected supernatant was incubated overnight at 4°C with 100 µl antimyosin II serum, after which it was incubated for 2 h at 4°C with 20 µl 50% protein A/G sepharose. The immune complexes bound to protein A/G were collected by centrifugation for 5 min at 12,000 g. The pellets were first washed in 1 ml lysis buffer and then once with a 1:1 dilution of lysis buffer/PBS, twice with PBS, and finally once with a 1:1 dilution of PBS/distilled water. The pellets were then boiled in Laemmli sample buffer (2% sodium dodecyl sulfate, 10% glycerol, 100 mM dithiothreitol, 60 mM Tris-HCl, pH 6.8, 0.001% bromphenol blue) and were subjected to 12.5–15% polyacrylamide gel electrophoresis. The separated proteins were electrophoretically transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore) and were blocked for 1 h with 10% skim milk in Tris-buffered saline (TBS) containing 0.2% Tween 20. The blots were probed with mouse anti-P-Ser Ab (7F12; 1:1000 dilution), mouse anti-P-Thr Ab (14B3; 1:1000 dilution), and mouse anti-MLC mAb (MY-21; 1:200 dilution). After washing five times with 0.2% Tween 20-TBS, blots were incubated for 1 h with horseradish peroxidase-conjugated goat anti-mouse polyclonal Ab (1:4000 dilution, Dako, Carpinteria, CA), washed again five times with 0.2% Tween 20-TBS, visualized by enhanced chemiluminescence (Amersham, Buckinghamshire, UK), and exposed to Fuji RX-U X-ray film, which was developed using a Konica automatic processor SRX-101. The 20-kDa protein immunoprecipitated by antimyosin II serum was considered to be MLC. Results were normalized to baseline optical densities (ODs) of bands containing phosphorylated endothelial MLC using NIH image software.
Enzyme inhibition
To determine the effect of inhibiting Rho, Rho kinase, and MLCK, HUVECs on glutaraldehyde cross-linked gelatin or polycarbonate membranes were preincubated at 37°C with a Rho inhibitor, C3 transferase (5 or 10 mg/ml; 24 h); a Rho-kinase inhibitor, Y-27632 (5 or 10 µM; 30 min); or a MLCK inhibitor, ML-7 (30 µM; 30 min). After washing three times with PBS, transendothelial neutrophil migration, F-actin staining, myosin II staining, and myosin immunoprecipitation were assessed.
Statistics
Values are expressed as the means ± SD. The significance of differences between groups was assessed by one-way ANOVA with Scheffe’s multiple comparison tests. Values of P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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With the exception of the low dose (5 mg/ml) of C3 transferase, pretreatment with any of the aforementioned inhibitors significantly diminished migration of neutrophils across HUVEC monolayers (Fig. 2a) . Conversely, there was no significant difference with the number of neutrophil adhesion to endothelial cells between control monolayers and those pretreated with an inhibitor (Fig. 2b) . This results were consistent with several reports [12 , 13 ], and we speculated that many neutrophils that had adhered to endothelial cells but were unable to migrate across endothelial cell monolayers might have detached, thus accounting for the smaller number of neutrophils associated with inhibitors treated with HUVEC monolayers in this migration assay. By contrast, pretreatment of the collagen gel matrix in the absence of endothelial cells did not affect neutrophil migration (Fig. 2c) , which confirms that the diminished migration across pretreated monolayers cannot be attributed to an inhibitor remaining in the transwell chamber after washing with PBS. These findings suggest that endothelial Rho and Rho kinase signaling is involved in the process of transendothelial neutrophil migration.
F-actin and myosin II reorganization during transendothelial migration of neutrophils
The observed neutrophil migration was associated with endothelial actin polymerization and myosin II reorganization. At the time transendothelial neutrophil migration was initiated by exposure to10-7 M LTB4, a rim of F-actin staining could be seen at the margins of control cells, along with a few randomly oriented stress fibers within the cytoplasm (Fig. 3a
). Simultaneously, myosin II was diffusely distributed throughout the cytoplasm and exhibited no organized pattern (Fig. 3h)
. However, in the continued presence of LTB4, progressive actin polymerization (Fig. 3b
, 5 min;3c
, 15 min; 3d
, 30 min; 3e
, 60 min) and myosin II redistribution (Fig. 3i
, 5 min; 3j
, 10 min; 3k
, 30 min; 3l
, 60 min) resulted in formation of organized filamentous networks. Neutrophils alone and LTB4 alone caused a slight increase in actin and myosin II filaments (Fig. 3f
and 3m
, neutrophil alone; Fig. 3g
and 3n
, LTB4 alone), but those were almost identical in control cells (Fig. 3a
and 3h)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The bacterial toxin Clostridium botulinum exoenzyme C3 transferase specifically inhibits the small G-proteins of the Rho subfamily by ADP-ribosylating an asparagine (Asn-41) at the GTP-binding site [28 29 30 ]. In our experiments, we used C3 transferase at concentrations of 5 or 10 mg/ml (24 h incubation), which were chosen because at those concentrations, C3 transferase selectively inhibits Rho-mediated, thrombin-induced endothelial cell contraction [25 ]. The effects of C3 transferase on cellular function may be limited by its slow uptake into cells, which is attributed to inefficient endocytosis by the stress fiber-containing subpopulation and results in a heterogeneous F-action stress fiber morphology [38 ]. In the present study, C3 transferase-treated HUVEC monolayers displayed similar heterogeneous F-actin morphologies; nonetheless, all C3 transferase-treated HUVECs exhibited diffuse myosin II staining and significant inhibition of MLC phosphorylation. It is interesting that the homogeneity of the myosin II morphology suggests that C3 transferase had a greater effect on formation of myosin II filaments than F-action stress fibers.
Y-27632 is a relatively selective inhibitor of Rho kinase. In vitro, Y-27632 inhibits Rho kinase with a Ki = 0.14 µM, which is about 185 times lower than that for protein kinase C (Ki=26 µM); Y-27632 does not significantly inhibit MLCK (Ki>250 µM). At a concentration of 10 µM, Y-27632 maximally inhibits Rho kinase-dependent cell contraction, and at concentrations of 5–10 µM, it also selectively inhibits Rho kinase-dependent effects in other cell types [32 , 39 ]. That the same concentration of Y-27632 inhibited endothelial actin polymerization, myosin II rearrangement, MLC phosphorylation, and transendothelial neutrophil migration is strongly indicative of the key role played by Rho kinase in those processes.
Cui et al. [40 ] reported that LTB4-induced neutrophil chemotactic migration across confluent endothelium results in activation of endothelial phospholipase D, and endothelial challenge with LTB4 had no effect [13 , 14 , 40 ]. LTB4 (10-7 M) was used as a chemoattractant in our experiment, as this concentration induced the highest rate of neutrophil migration [13 ]. We have made similar observations with formyl-Met-Leu-Phe (fMLP; data not shown), and fMLP-induced neutrophil migration is known to be associated with a rise in endothelial cytosolic-free calcium [12 ] and contraction [18 ]. Whether other chemotactic mediators behave the same way is unknown.
Thrombin not only induces endothelial cell contraction via Rho-Rho kinase-dependent inactivation MLC phosphatase [25
], it also induces increases in intracellular-free calcium, leading to activation of calcium/calmodulin-dependent MLCK, which phosphorylates MLC [41
] on serine 19 [18
]. Conversely, intracellular calcium gradients within endothelial cells are known to be coupled to transendothelial neutrophil migration across cytokine (interleukin-1 or tumor necrosis factor 
)-activated endothelial cells grown on human amnion preparations [12
]. In that case, neutrophil adhesion to endothelial cells induced transient increases in cytosolic calcium that paralleled the time course of neutrophil transmigration. Morever, this is consistent with our earlier report that endothelial MLC phosphorylation catalyzed by calcium/calmodulin-dependent MLCK regulates transendothelial neutrophil migration [13
]. Taken together, the aforementioned findings provide clear evidence that transendothelial neutrophil migration is regulated by endothelial cell-dependent cytoskeletal mechanisms and that a variety of intracellular signaling steps contribute to that process. Furthermore, the present results represent the first demonstration that chemoattractant-stimulated neutrophils induce serine and threonine phosphorylation of MLC via a small G-protein (Rho)-Rho kinase pathway and clarify part of the endothelial signal transduction mechanism underlying transendothelial neutrophil migration.
Although several adhesion molecules expressed on neutrophils (ß1 and ß2 integrins, refs. [4 5 6 7 ], CD47, ref. [42 ]) and endothelium (ICAM-1, refs. [6 7 8 ], PECAM-1, refs. [9 10 11 ]) have been implicated in transendothelial neutrophil migration, the actual trigger on the endothelial cells for neutrophil transmigration is still unknown. We speculate that when they adhere to endothelial cells, neutrophils may release signaling molecules that activate endothelial calcium/calmodulin-dependent MLCK and Rho. Rho in turn stimulates Rho kinase, which inactivates myosin phosphatase, which otherwise binds to phosphorylated MLC and dephosphorylates it. Rho kinase thus increases the level of MLC phosphorylation, which is cooperatively regulated positively by MLCK and negatively by a myosin phosphatase. In addition, neutrophil migration via MLC phosphorylation may be regulated not only by calcium/calmodulin-dependent MLCK but also by the Rho signaling pathway (Fig. 7 ).
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ACKNOWLEDGEMENTS |
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Received June 18, 2001; revised October 25, 2001; accepted October 29, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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