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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Physiology, College of Medicine,
National Cheng-Kung University, Tainan, Taiwan, Republic of China.
Vascular endothelium plays an important role in regulating
the transendothelial migration of polymorphonuclear leukocytes (PMNs).
In this study, the intracellular calcium ion
([Ca2+]i) signaling of endothelial cells
(ECs) during PMN transmigration was examined at the single-cell level.
Human umbilical vein ECs were cultured on a thin layer of collagen gel.
The ECs were labeled with fura-2, immersed in formyl-Met-Leu-Phe, and
subsequently perfused with fresh buffer to establish a gradient of
chemoattractant across the EC monolayer. The entire process of PMN
rolling on, adhering to, and transmigrating across the EC monolayer was
recorded under both phase-contrast and fluorescence optics. The data
showed the following: (1) At high concentration (approximately
3 × 106/mL), both PMN suspension and its supernatant
stimulated frequent EC [Ca2+]i elevations
across the monolayer; (2) when used at lower concentration (approximately 5 × 105/mL) to avoid the interference of
soluble factors, PMN transmigration, but not rolling or adhesion, was
accompanied by EC [Ca2+]i elevation; (3) the
latter EC [Ca2+]i elevation occurred
simultaneously in ECs adjacent to the transmigration site, but not in
those that were not in direct contact with the transmigrating PMNs; (4)
this EC [Ca2+]i elevation was an
initial and required event for PMN transmigration; and (5) PMNs
pretreated with 5,5'-dimethyl-1,2-bis(2-aminophenoxy)ethane-N, N, N',
N'-tetraacetic acid transmigrated with the accompanying EC
[Ca2+]i elevation, but they became elongated
in the collagen gel. In conclusion, PMNs induce adjacent EC
[Ca2+]i signaling, which apparently mediates
the "gating" step for their subsequent transmigration.
(Blood. 2000;96:3816-3822) Intercellular junctions connecting a monolayer of
vascular endothelial cells (ECs) occur as a complex network of
transmembrane proteins. Presumably, they not only determine endothelial
permeability, but also control leukocyte
transmigration.1,2 One striking characteristic of the
endothelial junctions is their dynamic organization, which is mostly
quick and reversible; that is, the endothelium is able to disorganize
and reorganize its intercellular junctions within minutes. It is
conceivable that inflammatory agents or leukocytes bind to specific
receptors on the EC surface and generate intracellular signals, which
in turn cause cytoskeletal reorganization and opening of intercellular
junctions. However, the mechanism that governs these processes is
largely unknown at present.
One of the most intriguing features of EC-leukocyte interactions is
the endothelial intracellular cytosolic free calcium concentration ([Ca2+]i) signaling that influences
transmigration. When polymorphonuclear leukocytes (PMNs) are placed on
human umbilical vein EC monolayers cultured on an amnion that separates
chemoattractant, a [Ca2+]i rise occurs in
single ECs.3 This EC [Ca2+]i
rise is associated with PMN-EC interactions, including PMN adhesion
and transmigration. Moreover, ECs remain relatively quiescent when
exposed to PMNs in the absence of chemoattractant. According to another
report, a PMN adhesion-associated EC [Ca2+]i
elevation is observed when a chemoattractant-free PMN suspension flows
through a glass tube covered with a layer of human aortic ECs.4 In either study, large numbers of PMNs as well as
possible bioactive soluble factors encountered individual ECs within a short time. However, neither the exact PMN-EC contact time nor the
sequence of postcontact cellular events could be recorded.
In general, leukocyte adhesion and transmigration across the
endothelial monolayer is an important physiologic process that probably
involves EC [Ca2+]i signaling and the opening
of EC junctions. However, the detailed steps of leukocyte
transendothelial migration remain obscure, mainly because of technical
difficulties. We report the development of a suitable model system to
monitor this complex process step by step and to investigate the
possible role of EC [Ca2+]i signaling in PMN
transmigration. We cultured human umbilical vein ECs on a thin layer of
collagen gel until confluence. The EC monolayer was labeled with
fura-2, immersed in formyl-Met-Leu-Phe (fMLP), and subsequently
perfused with fresh buffer to establish a gradient of chemoattractant
across this EC monolayer. The entire process of PMN rolling on,
adhering to, and transmigrating across the EC monolayer was examined
under phase-contrast optics. The fluorescence ratio images were
simultaneously recorded to monitor the EC
[Ca2+]i level. At the end of experimentation,
the specimen was fixed and further processed for observation under
either a confocal or a scanning electron microscope.
Materials
EC isolation and culture
PMN isolation PMNs were isolated from human peripheral blood by centrifugation on a discontinuous Ficoll-Hypaque gradient, according to a previous report.5 Purified PMNs were resuspended in Krebs-Ringer HEPES (KRH) buffer (125 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L KH2PO4, 1 mmol/L MgSO4, 2 mmol/L CaCl2, 25 mmol/L HEPES, 6 mmol/L glucose, and 10% fetal bovine serum, pH 7.4) and held on ice for no more than 3 hours before use. In some experiments, PMN sedimentation was carried out for 1 hour at 4°C under gravitational force, and thereafter the supernatant was used to test the possible effect of soluble factors in our transmigration model system.Chemoattractant gradient formation and endothelial [Ca2+]i measurements The confluent EC monolayer grown on collagen gel was replenished with KRH buffer and allowed to stand overnight. The specimen was then immersed in 1 µmol/L fMLP for 1 hour. After this step, EC-treating reagents (fura-2 AM and BAPTA-AM) were prepared in the same fMLP-containing buffer. ECs were labeled for 40 minutes at room temperature with 5 µmol/L fura-2 AM, the fluorescent Ca2+ indicator. Extracellular fura-2 AM was washed away, and the EC monolayer was incubated for an additional 20 minutes at 37°C in fura-2-free buffer. In some experiments, the specimen was incubated for 25 minutes with 50 µmol/L BAPTA-AM at 37°C to block the EC [Ca2+]i elevation. The cover glass containing the labeled specimen was assembled on a flow chamber6,7 that was slightly modified to accommodate the extra thickness of collagen gel. The chamber was mounted on an inverted microscope (Diaphot 300; Nikon, Tokyo, Japan) and was continuously perfused with fMLP-free KRH buffer (0.05 mL/min, 37°C) to wash away fMLP from the EC apical surface. Thus, a gradient of fMLP was established across the EC monolayer.The experimental setup for endothelial [Ca2+]i imaging was similar to the one used for [Ca2+]i measurements in single platelets and single ECs.8,9 After the flow chamber had been perfused with fMLP-free KRH buffer for 5 minutes (0.05 mL/min), PMNs were added to the flow chamber at the same flow rate. Phase-contrast and fluorescence images were recorded intermittently to monitor the PMN transmigration process and the accompanying EC [Ca2+]i changes, respectively. All experiments were performed at 37°C. At the end of some experiments, the calcium concentration was calibrated by applying ionomycin (5 µmol/L) in the presence of 5 mmol/L EGTA, followed by 10 mmol/L CaCl2. All signals were corrected for autofluorescence determined by exposing the tissue to 5 mmol/L manganese to quench cytosol fura-2 fluorescence. After subtracting the background and the autofluorescence, endothelial [Ca2+]i was estimated according to an established formula.10 Because this calibration procedure damages the cell morphology, it was performed only in some experiments to demonstrate the magnitude of the EC [Ca2+]i elevation. At the end of most experiments, the specimens were fixed with glutaraldehyde (2.5%) and stained with silver nitrate11 to visualize the endothelial boundary. Confocal microscopy and scanning electron microscopy For confocal microscopy, fixed cells were permeabilized with 0.5% Triton X-100 and fluorescently labeled with Alexa Fluor-488-conjugated phalloidin. A field containing the PMN transmigration site was focused under fluorescence optics, and the optic sections of it were examined under the confocal microscope (MRC 1000; BioRad, Hemel Hempstead, Hertfordshire, England). For scanning electron microscopy, nonpermeabilized specimens were further fixed in 1% OsO4, 1% tannic acid, and saturated uranyl acetate to minimize handling artifacts.12 The specimens were subsequently dehydrated in increasing concentrations of acetone, critical-point dried, and coated with gold. Transmigrating PMNs that were previously recorded under a light microscope were identified and were further examined under a scanning electron microscope (S 2500; Hitachi, Tokyo, Japan).
Our preliminary experiments showed that PMNs could migrate into a fMLP-treated collagen gel even in the absence of the EC monolayer (data not shown). When the EC/collagen layer was not pretreated with fMLP, PMNs migrated on the EC monolayer and maintained a relatively round shape for long periods, and were easily flushed away under flow. In contrast, they underwent rapid transendothelial migration through an fMLP-pretreated EC/collagen layer. However, this chemoattractant gradient was present only when fMLP-free buffer was continuously flowing through the chamber. Upon cessation of flow, PMN transmigration soon stopped, which indicates that the chemoattractant gradient was abolished. When the flow was reinitiated, PMNs resumed their transmigration. This process could be repeated several times until the ultimate depletion of fMLP. The morphology of transmigrating and transmigrated PMNs is shown in
Figure 1. Under phase-contrast light
microscopy, transmigrating cells usually showed bright cell bodies and
dark, irregular protrusions (Figure 1A). Their cell bodies, but not
protrusions, were also visible under the scanning electron microscope
(Figure 1B-C). Visibility of both PMN protrusions and collagen fibers
underneath the EC layer was noted only when an occasional gap was
present near the transmigration site (scanning electron micrograph not shown). Whereas transmigrated cells were dark and irregular under phase
contrast (Figure 1A), they were almost invisible in scanning electron
micrographs (Figure 1B). When scanning electron micrographs and
corresponding phase-contrast images were carefully examined, the
locations of PMN transmigrated sites appeared as slightly raised areas
(Figure 1B,D).
With silver staining, the EC boundary became clearly visible under
bright-field optics (Figure 2A). PMNs
were found to migrate preferentially to the tricellular corners,
confirming previously published results.11 Moreover,
compared with PMNs located elsewhere, PMNs located at the tricellular
corners were more resistant to flow-induced detachment. Although PMNs
were not fluorescently labeled and hence were invisible under
fluorescence microscopy, their locations could be identified by mapping
the fluorescent image with the corresponding silver-stained image
(Figure 2B).
To examine the possible effect of soluble factors under our
experimental conditions, we compared PMN suspension-evoked and supernatant-evoked EC [Ca2+]i signaling. At a
concentration of 3 × 106 cells/mL, both the suspension
and its supernatant were able to induce EC
[Ca2+]i elevation (Figure
3A-B). Although the average
[Ca2+]i elevation of these ECs was low, many
individual ECs showed [Ca2+]i spikes as high
as 500 nmol/L. However, it was difficult to distinguish the
cell-mediated effects from those mediated by soluble factors. To
overcome this problem, we reduced the concentration of the PMN
suspension to 5 × 105 cells/mL. ECs were relatively
quiescent when exposed to the resulting supernatant (Figure 3C).
Therefore, a PMN concentration of 5 × 105 cells/mL was
used for further studies.
The PMN transmigration and EC [Ca2+]i
signaling were monitored simultaneously under phase-contrast video
recording and ratio imaging of fura-2 fluorescence, respectively. When
signals from an area covering more than 50 ECs were averaged, there was
no observable EC [Ca2+]i elevation upon the
exposure of a PMN suspension. At the single-cell level, although
occasional [Ca2+]i spikes were observed in
some ECs, the [Ca2+]i level in most ECs
remained quiescent (usually less than 100 nmol/L) throughout the
experiment. Moreover, EC [Ca2+]i elevation
did not occur when PMNs were in contact with, rolling on, or adhering
to the ECs, which all happened in the couple of minutes between PMN
arrival and transmigration (Figure 4). On the other hand, the [Ca2+]i elevation of ECs
adjacent to the PMNs occurred simultaneously with PMN transmigration.
Concomitant [Ca2+]i elevations in ECs that
were one cell away from the PMN transmigration site were never
observed. Thus, the [Ca2+]i elevation did not
occur in any other EC that was not in direct contact with the
transmigrating PMN. Consistent with the fact that PMNs preferentially
located at tricellular corners in the EC monolayer (Figure 2), the
majority of PMNs transmigrated with more than one concomitant EC
[Ca2+]i signal. Among 79 transmigrated PMNs,
15 were accompanied by no concomitant EC
[Ca2+]i elevation, 6 by 1 EC
[Ca2+]i elevation, 20 by 2 EC
[Ca2+]i elevations, 34 by 3 EC
[Ca2+]i elevations, and 4 by 4 EC
[Ca2+]i elevations. Those PMNs that
transmigrated without accompanying EC [Ca2+]i
elevations probably went through the EC monolayer via interendothelial gaps, which would be present in cultured cell monolayers.
It is interesting to note that only ECs adjacent to the transmigration site, but not the ECs even one cell away, showed [Ca2+]i elevation (Figure 4). Transmigration-induced EC [Ca2+]i elevation was thus a local phenomenon that did not spread to other ECs of the same monolayer. However, this EC [Ca2+]i elevation did not seem to be subcellularly localized, ie, it happened throughout the entire cell. When images of these transmigration-affected ECs were divided into halves, one being adjacent to the transmigration site (proximal half) and the other being away from it (distal half), the responses were identical between the halves (data not shown). Because of the limited time resolution (a few seconds) of our imaging setup, we were unable to detect the possible [Ca2+]i wave propagation within single endothelial cells. To investigate at which stage of PMN transmigration the EC
[Ca2+]i signaling actually happens, we fixed
the specimens immediately after the occurrence of EC
[Ca2+]i elevations. Figure
5 shows an example. The fixative was in contact with the ECs within 20 seconds after the initiation of EC
[Ca2+]i signaling, and the major portion of
the cell body of this transmigrating PMN was still above the EC
monolayer (confocal images in Figure 5). Thus, it is clear that EC
[Ca2+]i signaling happens at an early stage
of PMN transmigration.
Whether EC [Ca2+]i signaling is required for
PMN transmigration or is merely an affiliated effect is an interesting
question. The EC [Ca2+]i signaling could be
blocked by pretreatment with the cell-permeable [Ca2+]i chelator BAPTA-AM, as indicated by
the complete absence of ratio changes in fura-2 fluorescence (data not
shown). Treatment of ECs with 50 µmol/L BAPTA-AM was sufficient to
block both EC [Ca2+]i elevation and PMN
transmigration under a fMLP gradient. Under these conditions, PMNs
formed protrusions that extended underneath the EC monolayer (Figure
6). Although these protrusions
continuously changed their shape and direction, the cell bodies
remained round above the monolayer for about 1 hour. Treatment of ECs
with higher concentrations of BAPTA-AM caused obvious gaps in the
monolayer without blocking PMN transmigration. These results confirm
the previous finding that PMNs remain on top of a monolayer of ECs whose [Ca2+]i has been clamped with
intracellular calcium chelators.3
PMN [Ca2+]i elevations have been demonstrated
during their migration.13 To determine whether PMN
[Ca2+]i elevation was a prerequisite for EC
[Ca2+]i signaling and their own
transendothelial migration, we pretreated PMNs with 50 µmol/L
BAPTA-AM. Treatment with this concentration of BAPTA was enough to
prevent even fMLP-evoked PMN [Ca2+]i
elevation (data not shown). [Ca2+]i-buffered
PMNs were capable of migrating on the EC monolayer and inducing normal
EC [Ca2+]i signaling, but their morphology
was drastically altered after transendothelial migration (Figure
7). Among 23 transmigrated PMNs, 5 were
accompanied by no concomitant EC [Ca2+]i
elevation, 2 by 1 EC [Ca2+]i elevation, 6 by
2 EC [Ca2+]i elevations, and 10 by 3 EC
[Ca2+]i elevations. Although entire cell
bodies penetrated the collagen gel, these
[Ca2+]i-buffered PMNs often stretched more
than 30 µm, with rear ends located just underneath the EC monolayer.
As a comparison, 377 of 388 transmigrated normal PMNs were shorter than
25 µm, but 295 of 346 BAPTA-treated PMNs that had already
transmigrated were longer than 25 µm.
In this study, an in vitro flow system has been developed to examine the entire process of PMN rolling, adhesion, and transendothelial migration, along with the accompanying EC [Ca2+]i signaling events. Our results demonstrate that PMN transmigration across the EC monolayer, but not rolling or adhesion, is accompanied by [Ca2+]i signaling in the ECs adjacent to the transmigration site. Furthermore, this EC [Ca2+]i signaling happens at an early stage of PMN transmigration and is essential for the PMN cell body to pass through. Although the underlying molecular mechanism that triggers the EC [Ca2+]i signaling is unknown at present, it seems that direct contact between the transmigrating PMN and the adjacent ECs is needed for this triggering event. There are several reasons that support this point of view. First, EC [Ca2+]i signaling happens only in those ECs in contact with a transmigrating PMN. Second, if some soluble factors served as the trigger instead, one would observe [Ca2+]i elevation preferentially happening in the particular EC downstream of the PMN transmigration site. However, we did not observe such a phenomenon. Finally, PMNs pretreated with BAPTA, presumably with their secretion machinery blocked, were able to induce normal EC [Ca2+]i signaling. This is consistent with previous findings that neither the rapid rise in PMN [Ca2+]i nor specific granule fusion with the plasma membrane constitutes a prerequisite for PMN migration across the EC monolayer.14 We have tested a more concentrated PMN suspension (3 × 106 cells/mL) in our flow chamber system. Arrival of the PMN suspension caused random [Ca2+]i elevations in many ECs and resulting significant [Ca2+]i elevation of the entire EC monolayer (Figure 3A), which is similar to a previous report in which approximately 5 to 10 PMNs per EC in the monolayer were used.4 [Ca2+]i elevation in the EC monolayer under the latter experimental conditions happened at the moment of suspension arrival and was not restricted to ECs adjacent to the transmigration sites. Our data showed that similar EC [Ca2+]i elevations could be generated by the supernatant isolated from 3 × 106 cells/mL PMN suspension (Figure 3B). In our hands, the supernatant from a fMLP-pretreated PMN suspension induced an EC [Ca2+]i elevation whose cross-monolayer average value could reach 500 nmol/L (data not shown). Therefore, some soluble factors released from concentrated PMNs, regardless of whether these cells have been treated with chemoattractant, may be responsible for the immediate cross-monolayer EC [Ca2+]i elevation that is independent of PMN transmigration.3,4 As a comparison, clear adhesion-associated EC [Ca2+]i signaling has been established when natural killer (NK) cells, but not other lymphocytes, adhere to cultured human umbilical vein ECs.15 In one example reported in that study, simultaneous recording of NK cells and ECs at the single-cell level revealed that EC [Ca2+]i elevation happened shortly (50 seconds) after [Ca2+]i elevation in adherent NK cells. Moreover, the NK [Ca2+]i elevation was a prerequisite for the adhesion-dependent EC signaling. Although it is possible that some secreted products may be involved, blockage of the NK secretion pathway was unable to prevent [Ca2+]i elevation in either cell type. Together with our current results, the data indicate that contact-mediated outside-in signaling in ECs may be present in PMN transmigration across an EC monolayer as well as in NK cell EC adhesion. Because the PMN transmigration-associated [Ca2+]i signaling spread across the entire EC cytoplasm, some other unidentified signaling/response components must be localized subcellularly to serve as a relay between [Ca2+]i signaling and intercellular junction opening. Phosphorylation of myosin light chains could serve as a [Ca2+]i-dependent relay because it appears to be a critical event in PMN-induced EC cytoskeletal alterations that accompany EC contractility.16 Activation of myosin light chain kinase (MLCK) is a well-known Ca2+/calmodulin-dependent event. MLCK can also regulate agonist- and flow-stimulated Ca2+ influx in ECs.17 Leukocytes may regulate this enzyme activity in ECs through the induction of postcontact EC [Ca2+]i signaling, thereby promoting their transmigration. It has also been shown that either Ca2+ chelators or calmodulin/MLCK inhibitors effectively block PMN transmigration without inhibiting PMN adhesion.18 This is consistent with our observation that EC [Ca2+]i signaling appears to be related to PMN transmigration but not to adhesion. It would be interesting to examine the subcellular localization of MLCK in ECs. Of the several types of junctional complexes identified at the EC boundary, the adherens junction seems to be the primary candidate in mediating transendothelial migration. Whether transendothelial migration requires the disassembly of adherens junctions appears to depend on the type of transmigrating cells. For example, ECs have been reported to actively participate in the transmigration of melanoma cells by disassembling and reassembling VE-cadherin-rich adherens junctions.19 However, adherens junctions remain intact during diapedesis of monocytes.20 Tight junctions, on the other hand, are unlikely to be involved because they are absent at tricellular corners.11 Whether PMN transmigration is accompanied by the breakdown of endothelial barrier function is an important issue. Although PMNs may stay on the EC monolayer for minutes, the transmigration process of individual PMNs is completed in less than a minute, as indicated by video images recorded under phase-contrast optics. The present results are consistent with those reported previously.21 We have never observed any physical gaps between a transmigrating PMN and the ECs in contact with it. This indicates that PMNs probably find their way through the transmigration sites that are preferentially located at tricellular corners. In contrast, interendothelial gaps caused by histamine treatment, which mostly occurred between 2 adjacent ECs, have been regarded as sites for plasma leakage in inflammation.22 These gaps last for hours and show complex morphology, such as fingerlike cell processes. The current consensus is that leukocyte transmigration requires mechanisms that open EC junctions to allow leukocyte passage. However, it is controversial whether the leukocyte adhesion step alone can account for such mechanisms. Recently, it has been suggested that PMN adhesion, not the subsequent transmigration, triggers the disorganization of endothelial junctions.23,24 This point of view has been challenged by evidence that some methodologic artifacts may cause disappearance of the EC adherens junctional complex.25 PMN constituents, especially proteolytic enzymes, are released during detergent lysis of these cells. A high local concentration of these proteolytic enzymes could lead to specific degradation of the EC junctional complex, even with protease inhibitors in the bulk phase. PMN [Ca2+]i elevation seems to be required for PMN detachment from the collagen gel, but not from the EC surface. In this study, [Ca2+]i-buffered PMNs showed elongated shapes after transendothelial migration, apparently because of "sticky" rear ends (Figure 7). Close observation reveals that these rear ends were not on the EC apical surface. Instead, they were located in the collagen gel underneath the ECs adjacent to PMN transmigration sites. It has been reported that repeated transient increases in PMN [Ca2+]i are required for PMN migration either on 2-dimensional surfaces (coated with fibronectin or vitronectin) or through 3-dimensional matrices (human amnion).26,27 Moreover, RGD-containing peptides and antibodies that block integrin receptors are able to rescue the migration of [Ca2+]i-buffered PMNs. Therefore, these repeated [Ca2+]i increases are required for integrin-mediated PMN detachment from these surfaces or matrices. Because we observed that [Ca2+]i-buffered PMNs adhered to and migrated normally on ECs, RGD-dependent integrins are probably not involved in these PMN-EC interactions. In conclusion, we have provided additional evidence in support of the following model for PMN transmigration. Initially, PMNs form proximal protrusions that send out contact-mediated signals to activate adjacent EC [Ca2+]i signaling, which is required for subsequent opening of the intercellular junctions. The protrusions then migrate into the collagen gel and eventually pull the cell body through. Once initiated, the whole process usually takes less than l minute to complete. This model is consistent with previously reported results.3,21
We are grateful to Dr S. T. Jiang for his help in the development of the chemoattractant gradient system and to Dr H. Essackjee for his critical reading of this manuscript.
Submitted February 21, 2000; accepted August 10, 2000.
Supported by grants from The National Science Council and The National Health Research Institute, Taiwan, R.O.C. (grant numbers NSC 89-2320-B-006-034, NSC 89-2320-B-006-045, and NHRI-GT-EX89S834L).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Chauying J. Jen, Department of Physiology, College of Medicine, National Cheng-Kung University, Tainan 701, Taiwan, Republic of China; e-mail: jen{at}mail.ncku.edu.tw.
1. Bianchi E, Bender JR, Blasi F, Pardi R. Through and beyond the wall: late steps in leukocyte transendothelial migration. Immunol Today. 1997;18:586-591[Medline] [Order article via Infotrieve]. 2. Lampugnani MG, Dejana E. Interendothelial junctions: structure, signaling and functional roles. Curr Opin Cell Biol. 1997;9:674-682[Medline] [Order article via Infotrieve].
3.
Huang AJ, Manning JE, Bandak TM, Ratau MC, Hanser KR, Silverstein SC.
Endothelial cell cytosolic free calcium regulates neutrophil migration across monolayers of endothelial cells.
J Cell Biol.
1993;120:1371-1380
4.
Ziegelstein RC, Corda S, Pili R, et al.
Initial contact and subsequent adhesion of human neutrophils or monocytes to human aortic endothelial cells releases an endothelial intracellular calcium store.
Circulation.
1994;90:1899-1907 5. English D, Anderson BR. Single step separation of red blood cells, granulocytes and mononuclear leukocytes on discontinuous density gradients of Ficoll-Hypaque. J Immunol Methods. 1974;5:249-252[Medline] [Order article via Infotrieve].
6.
Jen CJ, Lin JS.
Direct observation of platelet adhesion onto fibrinogen- and fibrin-coated surfaces.
Am J Physiol.
1991;261:H1457-H1463 7. Jen CJ, Tai YW. Morphological study of platelet adhesion dynamics under whole blood flow conditions. Platelets. 1992;3:145-153.
8.
Jen CJ, Chen HI, Lai KC, Usami S.
Changes in cytosolic calcium concentrations and cell morphology in single platelets adhered to fibrinogen-coated surface under flow.
Blood.
1996;87:3775-3782
9.
Huang TY, Chu TF, Chen HI, Jen CJ.
Heterogeneity of [Ca2+]i signaling in intact rat aortic endothelium.
FASEB J.
2000;14:797-804
10.
Grynkiewicz G, Poenie M, Tsien RY.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J Biol Chem.
1985;260:3440-3450 11. Burns AR, Walker DC, Brown ES, et al. Neutrophil transendothelial migration is independent of tight junctions and occurs preferentially at tricellular corners. J Immunol. 1997;159:2893-2903[Abstract]. 12. Schroeter D, Spiess E, Paweletz N, Benke R. A procedure for rupture-free preparation of confluently grown monolayer cells for scanning electron microscopy. J Electron Microsc Tech. 1984;1:219-235. 13. Mandeville JTH, Maxifield FR. Calcium and signal transduction in granulocytes. Curr Opin Hematol. 1996;3:63-70[Medline] [Order article via Infotrieve]. 14. Kuijpers TW, Hoogerwerf M, Roos D. Neutrophil migration across monolayers of resting or cytokine-activated endothelial cells: role of intracellular calcium changes and fusion of specific granules with the plasma membrane. J Immunol. 1992;148:72-77[Abstract].
15.
Pfau S, Leitenberg D, Rinder H, Smith BR, Pardi R, Bender JR.
Lymphocyte adhesion-dependent calcium signaling in human endothelial cells.
J Cell Biol.
1995;128:969-978
16.
Hixenbaugh EA, Goeckeler ZM, Papaiya NN, Wysolmerski RB, Silverstein SC, Huang AJ.
Stimulated neutrophils induce myosin light chain phosphorylation and isometric tension in endothelial cells.
Am J Physiol.
1997;273:H981-H988
17.
Watanabe H, Takahashi R, Zhang XX, et al.
An essential role of myosin light-chain kinase in the regulation of agonist- and fluid flow-stimulated Ca2+ influx in endothelial cells.
FASEB J.
1998;12:341-348
18.
Saito H, Minamiya Y, Kitamura M, et al.
Endothelial myosin light chain kinase regulates neutrophil migration across human umbilical vein endothelial cell monolayer.
J Immunol.
1998;161:1533-1540 19. Sandig M, Voura EB, Kalnins VI, Siu CH. Role of cadherins in the transendothelial migration of melanoma cells in culture. Cell Motil Cytoskeleton. 1997;38:351-364[Medline] [Order article via Infotrieve]. 20. Sandig M, Negrou E, Rogers KA. Changes in the distribution of LFA-1, catenins, and F-actin during transendothelial migration of monocytes in culture. J Cell Sci. 1997;110:2807-2808[Abstract]. 21. Huang AJ, Furie MB, Nicholson SC, et al. Effects of human neutrophil chemotaxis across human endothelial cell monolayers on the permeability of these monolayers to ions and macromolecules. J Cell Physiol. 1988;135:355-366[Medline] [Order article via Infotrieve]. 22. McDonald DM, Thurston G, Baluk P. Endothelial gaps as sites for plasma leakage in inflammation. Microcirculation. 1999;6:7-22[Medline] [Order article via Infotrieve].
23.
Del Maschio A, Zanetti A, Corada M, et al.
Polymorphonuclear leukocyte adhesion triggers the disorganization of endothelial cell-to-cell adherens junctions.
J Cell Biol.
1996;135:497-510
24.
Allport JR, Ding H, Collins T, Gerritsen ME, Luscinskas FW.
Endothelial-dependent mechanisms regulate leukocyte transmigration: a process involving the proteasome and disruption of the VE-cadherin complex at endothelial cell-to-cell junctions.
J Exp Med.
1997;186:517-527
25.
Moll T, Dejana E, Vestweber D.
In vitro degradation of endothelial catenins by a neutrophil protease.
J Cell Biol.
1998;140:403-407
26.
Marks PW, Maxfield FR.
Transient increases in cytosolic free calcium appear to be required for the migration of adherent human neutrophils.
J Cell Biol.
1990;110:43-52 27. Mandeville JTH, Maxfield FR. Effects of buffering intracellular free calcium on neutrophil migration through three-dimensional matrices. J Cell Physiol. 1997;171:168-178[Medline] [Order article via Infotrieve].
© 2000 by The American Society of Hematology.
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  P. Alcaide, G. Newton, S. Auerbach, S. Sehrawat, T. N. Mayadas, D. E. Golan, P. Yacono, P. Vincent, A. Kowalczyk, and F. W. Luscinskas p120-Catenin regulates leukocyte transmigration through an effect on VE-cadherin phosphorylation Blood, October 1, 2008; 112(7): 2770 - 2779. [Abstract] [Full Text] [PDF]
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 W.-H. Su, H.-i. Chen, and C. J. Jen Polymorphonuclear leukocyte transverse migration induces rapid alterations in endothelial focal contacts J. Leukoc. Biol., September 1, 2007; 82(3): 542 - 550. [Abstract] [Full Text] [PDF]
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 W. M. Kuebler, K. Parthasarathi, J. Lindert, and J. Bhattacharya Real-time lung microscopy J Appl Physiol, March 1, 2007; 102(3): 1255 - 1264. [Abstract] [Full Text] [PDF]
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 S. L. Cuvelier, S. Paul, N. Shariat, P. Colarusso, and K. D. Patel Eosinophil adhesion under flow conditions activates mechanosensitive signaling pathways in human endothelial cells J. Exp. Med., September 19, 2005; 202(6): 865 - 876. [Abstract] [Full Text] [PDF]
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L. Liu, D. C. Cara, J. Kaur, E. Raharjo, S. C. Mullaly, J. Jongstra-Bilen, J. Jongstra, and P. Kubes LSP1 is an endothelial gatekeeper of leukocyte transendothelial migration J. Exp. Med., February 7, 2005; 201(3): 409 - 418. [Abstract] [Full Text] [PDF]
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 G. Cinamon, V. Shinder, R. Shamri, and R. Alon Chemoattractant Signals and {beta}2 Integrin Occupancy at Apical Endothelial Contacts Combine with Shear Stress Signals to Promote Transendothelial Neutrophil Migration J. Immunol., December 15, 2004; 173(12): 7282 - 7291. [Abstract] [Full Text] [PDF]
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K.-P. Chuang, Y.-F. Huang, Y.-L. Hsu, H.-S. Liu, H.-C. Chen, and C.-C. Shieh Ligation of lymphocyte function-associated antigen-1 on monocytes decreases very late antigen-4-mediated adhesion through a reactive oxygen species-dependent pathway Blood, December 15, 2004; 104(13): 4046 - 4053. [Abstract] [Full Text] [PDF]
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B. N. Stein, J. R. Gamble, S. M. Pitson, M. A. Vadas, and Y. Khew-Goodall Activation of Endothelial Extracellular Signal-Regulated Kinase Is Essential for Neutrophil Transmigration: Potential Involvement of a Soluble Neutrophil Factor in Endothelial Activation J. Immunol., December 1, 2003; 171(11): 6097 - 6104. [Abstract] [Full Text] [PDF]
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C. V. Carman, C.-D. Jun, A. Salas, and T. A. Springer Endothelial Cells Proactively Form Microvilli-Like Membrane Projections upon Intercellular Adhesion Molecule 1 Engagement of Leukocyte LFA-1 J. Immunol., December 1, 2003; 171(11): 6135 - 6144. [Abstract] [Full Text] [PDF]
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W.-H. Su, H.-i. Chen, and C. J. Jen Differential movements of VE-cadherin and PECAM-1 during transmigration of polymorphonuclear leukocytes through human umbilical vein endothelium Blood, November 15, 2002; 100(10): 3597 - 3603. [Abstract] [Full Text] [PDF]
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H. A. Edens, B. P. Levi, D. L. Jaye, S. Walsh, T. A. Reaves, J. R. Turner, A. Nusrat, and C. A. Parkos Neutrophil Transepithelial Migration: Evidence for Sequential, Contact-Dependent Signaling Events and Enhanced Paracellular Permeability Independent of Transjunctional Migration J. Immunol., July 1, 2002; 169(1): 476 - 486. [Abstract] [Full Text] [PDF]
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 R. O. Dull and J. G.N. Garcia Leukocyte-Induced Microvascular Permeability: How Contractile Tweaks Lead to Leaks Circ. Res., June 14, 2002; 90(11): 1143 - 1144. [Full Text] [PDF]
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S. K. Shaw, P. S. Bamba, B. N. Perkins, and F. W. Luscinskas Real-Time Imaging of Vascular Endothelial-Cadherin During Leukocyte Transmigration Across Endothelium J. Immunol., August 15, 2001; 167(4): 2323 - 2330. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
