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Prepublished online as a Blood First Edition Paper on December 5, 2002; DOI 10.1182/blood-2002-08-2396.
PHAGOCYTES
From the Division of Pulmonary, Allergy, and Critical
Care, Department of Medicine, University of Pennsylvania School of
Medicine, Philadelphia, PA.
Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), a
tyrosine phosphoprotein highly expressed on endothelial cells and
leukocytes, is an important component in the regulation of neutrophil
transendothelial migration. Engagement of endothelial PECAM-1
activates tyrosine phosphorylation events and evokes prolonged calcium
transients, while homophilic engagement of neutrophil PECAM-1 activates
leukocyte Although the mechanisms regulating initial cell
contact and adhesion have been well described,1 the
mechanisms underlying endothelial regulation of polymorphoneutrophil
(PMN) transmigration are not as well understood. It has been proposed
that neutrophil transmigration through endothelial cell (EC)
monolayers involves 2 distinct pathways. The first, called type I
transendothelial migration (TEM), requires direct neutrophil activation
with chemotactic agents such as
N-formyl-methionyl-leucinyl-phenylalanine (FMLP), leukotriene-B4 (LTB4), or interleukin-8
(IL-8). The second, type II TEM ("endothelium dependent"),
involves endothelial prestimulation by inflammatory mediators, such as
IL-1 One adhesion molecule implicated in neutrophil transmigration is
platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), a
130-kDa immunoglobulin (Ig) superfamily protein that is
expressed at high levels at cell-cell borders on endothelial
cells,7,8 as well as on leukocytes and
platelets.9 PECAM-1 has been implicated as a key regulator
of neutrophil transmigration in inflammation,10,11 ischemia reperfusion,12,13 and oxidant
injury.14 Interestingly, some evidence suggests that
PECAM-1 might selectively participate in TEM in the context of cytokine
(IL-1 There are a number of ways EC PECAM-1 could potentially regulate
TEM. Because regulation of PECAM-1-mediated TEM requires homophilic
(PECAM-1-PECAM-1) interactions between endothelial cell and neutrophil
PECAM-1,15,19,20 it was initially postulated that EC
PECAM-1 might function primarily as a cell adhesion molecule, creating
a haptotactic gradient that helped to guide neutrophils to endothelial
cell-cell borders.15 This hypothesis, which emphasizes the
importance of the characteristic cell-cell localization of PECAM-1, has
gained further credence by recent evidence suggesting that PECAM-1
functions as the apical component of a step-wise progression of cell
adhesion proteins, such as CD99, that regulate monocyte diapedesis
following the selectin- and integrin-mediated steps of tethering,
rolling, and adhesion.21 However, in both ECs and
leukocytes, PECAM-1 has also been found to have a substantial role in
activating and modifying signal transduction pathways,9 leading to the emergence of at least 4 other mechanistic possibilities.
One of the first signaling functions of PECAM-1 to be recognized was
its ability to activate Neutrophil transmigration has been shown to require increases in
intracellular EC calcium levels independent of PMN calcium signaling.27 Interestingly, homophilic PECAM-1 engagement
can activate prolonged calcium transients in human umbilical vein endothelial cells (HUVECs) and in an EC-like
mesothelioma-derived cell line (REN cells) transfected with human
PECAM-1 (RHP cells).28,29 Recently, EC PECAM-1 has also
been described as a key regulator of endothelial oxidant-activated
calcium signals, a function that may be critical to mediating EC-PMN
interactions.30 Thus, PECAM-1-dependent EC calcium
channel activation represents a third potential mechanism by which EC
PECAM-1 could regulate TEM.
Another potentially important feature of PECAM-1 is that the
cytoplasmic domain possesses a tyrosine-containing motif composed of 2 tandem SH2 binding sites (Tyr663/Tyr686) that can be
phosphorylated by Src and Csk family kinases.31,32 In ECs,
this domain may mediate association with the SH2-containing tyrosine
phosphatase SHP2,33,34 while leukocyte and platelet
PECAM-1 has been shown to associate with SHP1, PI3K, and other SH2
domain proteins.9,24,35,36 Thus, a fourth mechanism by
which EC PECAM could regulate transmigration would be that PECAM-1
engagement or oxidant exposure leads to phosphorylation of the SH2
domain-binding tyrosines on EC PECAM-1 leading to association with
SHP2 or other cytoplasmic proteins that in turn influence the
transmigration process. Finally, reports of other cytoplasmic proteins,
such as Genetic approaches to evaluate these alternatives have proved
impractical because of the high levels of constitutive PECAM-1 expression in ECs. We therefore used a previously described
PECAM-1-null "endothelial model" cell line, REN, that manifests
many phenotypic and signaling characteristics of ECs when stably
transfected or virally transduced with human
PECAM-1.28-30,39 Utilizing this EC model in a
transmigration assay based on standardized in vitro models of leukocyte
transmigration,5,40 we have defined the components of
PECAM-1-dependent neutrophil transmigration. We also report the
effects of specific PECAM-1 mutations known to disrupt monolayer
PECAM-1-dependent cationic signaling and/or cell border localization
on the regulation of PECAM-1-dependent neutrophil transmigration.
Antibodies and reagents
Cells and PECAM-1 mutant constructs
Neutrophil transmigration assay
Human neutrophils were obtained from volunteers following informed consent. PMNs were isolated by Ficoll gradient separation (Robbins Scientific, Sunnyvale, CA) followed by hypotonic red cell lysis. Cell viability was more than 95% by trypan blue dye exclusion following this methodology. Neutrophils were resuspended in RPMI media supplemented with 1% FBS (R1%), counted, and incubated at room temperature in blocking or control antibodies 20 minutes prior to onset of experiments. Monolayers (top well) were concurrently washed gently in phosphate-buffered saline (PBS) then incubated with antibodies in R1% for 20 minutes prior to assays. In some experiments bioactive anti-IL-8 serum45 or NI rabbit serum was added to top (luminal) or bottom (abluminal) wells at 1:200 prior to addition of PMN aliquots.46 Neutrophils (500 000 cells/well) were placed in the upper transwell chambers and transmigration was allowed to take place over 4 hours at 37°C. Cells were harvested from the top and bottom chambers with adherent cells gently washed and added to the respective top and bottom chamber aliquots. Media was aspirated and cells resuspended in 0.1 M K2PO4 (pH 7.0) solution. Myeloperoxidase (MPO) activity was detected following reaction in 0.083 mg/mL O-diansidine (ICN), Hanks buffered saline (with 0.25% bovine
serum albumin [BSA]), and 0.005% hydrogen peroxide. Reactions were terminated after 10 minutes with 0.1% sodium azide and
MPO activity measured as optical density at 460 nm.47 A minimum of three 500 000 cell aliquots were measured to yield the
maximal (total PMNs) reference standard for each experiment. MPO values
were compared with a standard curve performed with each assay ranging
from 37 500 to 1.5 million cells. Cell counts and assay conditions
were optimized to facilitate data acquisition from the linear portion
of the standard curve. Similar to results obtained in monocyte
transmigration,48 time-course experiments in
IL-1 Adhesion assay Cells were seeded on 12-well tissue-culture plates (Costar). Confluent monolayers were treated with either IL-1 (10 U/mL) or
vehicle for 24 hours; the media were then removed and cells were washed
with PBS. Cell monolayers were incubated with control or blocking
antibodies in R1% media for 30 minutes and identically treated PMN
aliquots were then added. After 30 minutes, nonadhering cells were
aspirated and monolayers gently washed with media. Wells were aspirated
dry, cells resuspended in 100 µL K2PO4, and MPO assays conducted as described in the previous paragraph.
PMN retention in transwell filters was measured by cutting out filters following transmigration (TM) experiments. Filters were then
agitated in 100 µL K2PO4 and MPO assays on
resultant supernatants conducted as previously described.
Immunofluorescence staining Cell monolayers were grown to confluence on transwell filters as described in "Neutrophil transmigration assay." Filters were washed in PBS, fixed with 3% paraformaldehyde for 20 minutes, and then permeabilized with iced 0.5% nonidet P-40 (NP-40) for 1 minute. After washing, fixed monolayers were stained with anti-PECAM-1 mAb 4G6 and counterstained with FITC-conjugated goat anti-mouse IgG as described.39 Cells were imaged on a Nikon inverted epifluorescence microscope with a × 40 fluorescence lens and a Nikon digital camera (both from Nikon, Tokyo, Japan).
Phenotypic and functional characteristics of REN and PECAM-1-transfected REN cells In order to use molecular approaches to directly investigate the mechanisms of PECAM-1-regulated neutrophil transmigration, we utilized a standard transwell chamber transmigration system using the endothelial-like REN cell line. REN cells do not express PECAM-1, but can be stably transfected with high levels of wild-type PECAM-1 that then localize to cell-cell borders, regulates calcium-signaling activity, and manifests tyrosine phosphorylation patterns similar to PECAM-1 in ECs.28-30,39 Like ECs, mesothelial-derived cells, such as REN, also express ICAM-1 and vascular cell adhesion molecule (VCAM), form cobblestone monolayers phenotypically similar to ECs, and can support cytokine-mediated neutrophil transmigration.28,44,49An important feature of EC monolayers is the differential expression of
adhesion molecules such as ICAM-1 and PECAM-1 in response to specific
cytokines and chemokines.21 Similar to ECs,50 REN and RHP cells manifested marked increases in ICAM-1
expression following exposure to TNF
Accordingly, REN and RHP cells were plated on transwell filters and
confluence was monitored by electrical resistance. Monolayer resistance
reached a plateau by days 4 to 6 of approximately 30 to 35
Similar to ECs, we found that basal (unstimulated) PMN transmigration
on REN and RHP monolayers was minimal, but could be markedly enhanced
either by creation of a chemotactic gradient (type I transmigration) or
by pre-exposure of the EC monolayer to cytokines, such as IL-1 As seen in Figure 2C, we found that proportionally, more than two
thirds of IL-1 Thus, with regard to transmembrane electrical resistance,
morphology, and cell and adhesion expression profiles, REN and RHP cells closely resemble EC monolayers. Interestingly, the presence of
PECAM-1 appears to contribute an ICAM-1-dependent component to
IL-1 Role of PECAM-1 in IL-1 pretreated REN and RHP monolayers in the setting of a
polyclonal anti-PECAM-1-blocking antibody (Houston) or a negative
control rabbit antibody directed against the highly expressed cell
surface protein, 2 microglobulin. In 4-hour TM experiments, anti-PECAM-1 antibody (Houston) inhibited PMN transmigration by 50%
(P < .05%) through RHP monolayers, compared with the
proportion of PMNs transmigrating while exposed to anti-2
microglubulin. Conversely, in REN cells, treatment of monolayers and
PMNs with either Houston or anti-2 microglubulin control yielded no
difference in transmigration (Figure 3A).
These findings indicate that IL-1-stimulated transmigration
consists of a PECAM-1-dependent (antibody-blockable) and a
PECAM-1-independent component (supported by REN or
antibody-blocked RHP monolayers) and that heterophilic interactions
between neutrophil PECAM-1 and non-PECAM-1 monolayer ligands do not
play a role in IL-1-stimulated PMN transmigration.
In order to utilize previously characterized reagents28,29
to further investigate the relationship of EC calcium signaling to
transmigration, as well as to facilitate experiments utilizing different antibody isotypes, TM experiments were conducted to directly
compare a panel of nonblocking controls with our anti- In order to choose the optimal anti-PECAM-1-blocking reagent,
antibody blockade studies were performed comparing the polyclonal antibody (Houston) with 3 monoclonal anti-PECAM-1 antibodies: mAb 62 (directed against Ig-like loop 1), mAb Hec 7 (directed against Ig-like
loops 1 and 2), and mAb 4G6 (directed against Ig-like loop 6).
Anti-PECAM-1 antibody blockade was dose dependent (with maximal effect
observed at 75-100 µg/mL; data not shown) and somewhat epitope
specific. As shown in Figure 3C, the polyclonal antibody was most
effective, although 2 of the monoclonal antibodies (mAb 62 and Hec7)
induced significant (P < .05) blockade of transmigration, confirming prior findings that PECAM-1 homophilic interaction domains
are important in mediating PECAM-1-regulated TEM.19 Interestingly, mAb 4G6, an antibody known to activate calcium signaling,28,29 did not significantly block TEM. None of
these anti-PECAM-1 antibodies blocked PMN transmigration in
IL-1 In order to assess the contribution of PECAM-1 expression to
IL-1 Finally, in order to confirm that results attributed to differences in
transmigration rates were not simply due to variations in cell
adhesion, we performed adhesion assays on cytokine-treated REN cell and
REN cell transfectant monolayers. As with ECs, where cytokine-induced
TEM is separable from adhesion,53 IL-1 Role of PECAM-1 in IL-8 and LTB4-mediated type I neutrophil transmigration Our findings clearly identified a PECAM-1-dependent component to IL-1-mediated type II (cytokine-induced) neutrophil transmigration. To determine if PECAM-1 played a role in type I TEM mediated by CXC
chemokines (IL-8) and leukotriene chemoattractants (LTB4), TM assays were conducted on REN and RHP monolayers. IL-8 (5 nm) or
LTB4 (100 nm) was added to the abluminal chamber at the
time of PMN addition (luminal) and results compared with TM through RHP
cell monolayers pretreated for 24 hours with IL-1. As shown in
Figure 4, PMN TM was markedly increased
after chemokine addition. Both REN and RHP supported identical
transmigration rates of up to 45% to 50% of total PMNs, levels
similar to unblocked TM through IL-1-stimulated RHP monolayers.
However, unlike the effect on IL-1-induced TM through RHP
monolayers, addition of anti-PECAM-1 antibody yielded no inhibition of
chemokine-induced transmigration through RHP or REN cells. Thus, in
contrast to IL-1-mediated transmigration, chemokine (IL-8) and
leukotriene (LTB4)-induced (type I) transmigration is
unaffected by the presence of PECAM-1.
Relationship of IL-8 (type I) transmigration and PECAM-1 in
IL-1 -stimulated
ECs and mesothelial cells may exceed 1 to 2 nm and underlie at least
part of cytokine-induced transmigration.45,46,54 In order
to determine the relationship between PECAM-1 and luminal (surface-expressed) or secreted (abluminal) IL-8 in the context of
IL-1-stimulated transmigration, we examined the effects of a
bioactive anti-IL-8 serum45 or negative control NI rabbit serum added to either the luminal (top) or abluminal (bottom) chamber
in conjunction with (luminal) anti-PECAM-1 (Houston) or anti-MHC-1
antibodies. As shown in Figure 5A, the
functional bioactivity of the anti-IL-8 serum was confirmed
as abluminal addition of anti-IL-8 serum completely blocked
IL-8-stimulated transmigration. As shown in Figure 5B, in
IL-1-treated RHP monolayers, baseline transmigration (50% of total
neutrophils added, column 1) was established in wells with control
antibodies on the luminal (anti-MHC-1) and abluminal surfaces
(NI serum). Similarly, wells treated with luminal anti-MHC-1 antibody
manifested no inhibition in PMN transmigration when coincubated with
luminal anti-IL-8 serum (67% total PMNs added, column 6) or luminal
NI serum (54% total PMNs, column 5). The lack of inhibition in
transmigration between columns 5 and 6 suggests that luminal surface
expression of IL-8 is not required for IL-1-mediated transmigration
as has been suggested in TNF -stimulated transmigration.50 Fluorescence cytometry analysis
of IL-1-treated RHP cells similarly revealed no surface IL-8
expression compared with untreated RHP cells (not shown).
In contrast, in IL-1 However, in anti-PECAM-1-treated (Houston) monolayers to which NI
rabbit serum was added to the abluminal side (column 3), up to a 59%
decrease in PMN migration was noted (compared with anti-MHC-1 control
in column 1), representing the PECAM-1 blockable component of
IL-1 Use of PECAM-1 mutant constructs to determine the mechanisms by which PECAM-1 regulates PMN transmigration Having established an endothelial-like model system in which we could demonstrate a clear PECAM-1-dependent component of IL-1-mediated transmigration, we used this system to address the
question of how monolayer or "endothelial" PECAM-1 regulates PMN transmigration.
To evaluate the role of PECAM-1-mediated EC signaling, we studied REN
cells stably transfected with a series of PECAM-1 mutants. The PITC
construct contains the extracellular domain of PECAM-1 fused to the
transmembrane and cytoplasmic domains of ICAM-1 (Figure 1). The ICAM-1
cytoplasmic region on this construct lacks the cytoplasmic
Tyr663/Tyr686 motif required for tyrosine phosphorylation and
disrupts PECAM-1-mediated cationic signaling, but supports homophilic
interaction and maintains cell border localization (not
shown).29,30,39 We hypothesized that if monolayer
PECAM-1-mediated cell signaling (due to either calcium flux or
cytoplasmic domain protein-protein interactions) were important in
transmigration, the PECAM-1-dependent component of
transmigration would be lost. As seen in Figure
6A, when transmigration assays on
IL-1
The preservation of a clear PECAM-1-dependent (antibody blockable) component in the absence of the PECAM-1 cytoplasmic and transmembrane domains (and their potential signaling and protein association functions) suggested that EC PECAM-1 serves primarily as an adhesion protein, either forming a haptotactic gradient (which would require PECAM-1 cell border localization) or functioning as a "passive" ligand for PMN PECAM-1. To directly address the role of PECAM-1 cell border localization and
determine whether a haptotactic gradient is required for
PECAM-1-dependent transmigration, we evaluated TM using 2 additional
REN cell lines stably transfected with mutant PECAM-1 constructs that
disrupt PECAM-1 cell border localization. The first, termed
PECAM-
Neutrophil transmigration through cytokine-stimulated endothelial monolayers is a complex process that requires cell adhesion and "adhesion-dependent" intracellular signaling followed by "adhesion-independent" calcium transients and other signal processes in both ECs and PMNs.27,55 The proteins regulating leukocyte TEM include an array of EC and PMN cell adhesion proteins including PECAM-1, ICAM-1 and -2, CD99, junctional adhesion molecule (JAM), integrin-associated protein (IAP), the CD11/18 integrins,4,10,21,51,56,57 and others. It is still not clear, however, how each of these proteins regulates diapedesis, particularly in the context of different transmigration stimuli. The focus of these experiments was to study the role played by "endothelial" PECAM-1 (in contrast to leukocyte PECAM-1) in neutrophil transmigration in response to specific stimuli and to define the role of monolayer PECAM-1 signaling versus ligand functions in this process. In our model of neutrophil transendothelial migration, REN cells
expressing full-length PECAM-1 were phenotypically and functionally similar to endothelial cells, supporting both type II (cytokine [IL-1 Consistent with reports that direct-PMN activators mediating type
I TEM (such as FMLP and IL-8) may not require
PECAM-1,16-18,57 we found that IL-8 and
LTB4 elicited equivalent TM rates through REN and RHP
monolayers that were unaffected by anti-PECAM-1 antibodies, confirming
that regulation by PECAM-1 is stimulus specific. Interestingly, we
found that cytokine (IL-1 Having defined the stimuli for and components of PECAM-1-dependent transmigration, we utilized REN cells stably transfected with mutant PECAM-1 isoforms known to selectively disrupt cell border localization, calcium signaling, and protein-protein association to directly address the mechanisms by which PECAM-1 regulates neutrophil transmigration. To our surprise, we found that neither PECAM-1 cell-cell border localization, PECAM-1-mediated calcium signaling, nor the cytoplasmic domain that supports tyrosine phosphorylation and SH2 protein association is absolutely required for PECAM-1-dependent regulation of PMN transmigration. The findings that PECAM-1-dependent leukocyte transmigration requires homophilic PECAM-1-PECAM-1 interaction (Nakada et al19 and Figure 3) and that the extracytoplasmic domain is sufficient to reconstitute PECAM-1-mediated TEM (Figure 6) suggest that the primary function of endothelial cell PECAM-1 is to serve as a "passive" ligand for neutrophil PECAM-1. Mechanistically, a broad range of stimuli such as crosslinking of
neutrophil PECAM-1 or CD98,22,24,25,58 as well as direct PMN activators such as PAF and FMLP,59 have been found to
activate leukocyte Interestingly, although PECAM-1-dependent transmigration appears to be
ICAM-1 dependent (Figure 2C), absolute transmigration rates (not shown)
and proportional levels of PECAM-1 involvement were identical in cell
lines with large differences in ICAM-1 expression such as PECAM- Evaluation of PECAM-1-null mice similarly demonstrates both the
subtleties and redundancy of the mechanisms underlying leukocyte TEM.
Studies evaluating PMN TEM in IL-1 Although REN cells appear to be an excellent model for ECs, they do have some differences from ECs that must be considered. For example, REN cells do not express selectins and although they express cadherins at their cell-cell junctions, they have N-cadherin rather than VE-cadherin (data not shown). We do not think that these differences affect our conclusions; however, they could be potentially more important in flow-based systems in which selectins might be more involved. Similarly, contrary to prior findings linking loop 6 epitopes in monocyte TEM through the basement membrane,20 we found no significant blockade of neutrophil transmigration with an anti-loop 6 antibody in this model system. Although this could be due to differences between leukocyte types studied (which, like monocytes and lymphocytes, may have different levels of basal TM or surface protein expression from PMNs64) or due to differences in model systems used, this would not alter our conclusions regarding PECAM-1-dependent transmigration mediated by homophilic PECAM-1 interactions between ECs and PMNs. We are in the process of confirming our observations using ECs derived from PECAM knock-out mice. In summary, we have used an endothelial model system to study the
structural requirements of the PECAM-1 molecule for regulation of
cytokine-activated neutrophil transmigration. Contrary to our initial
hypothesis, neither monolayer (endothelial) PECAM-1-regulated calcium
signaling nor cytoplasmic domain tyrosine phosphorylation events (or
protein-protein association) were required for PECAM-dependent TM.
Similarly, PECAM-1 cell border localization was not necessary for
PECAM-1-dependent TM (Figures 1,6). These findings suggest that monolayer (endothelial) PECAM-1 serves as a passive ligand for
neutrophil PECAM-1 that then functions as an active signaling receptor
in an ICAM-1-dependent manner. We also confirm that PECAM-1-dependent TM is stimulus specific because chemokine (IL-8)- and chemoattractant (LTB4)-mediated TEM does not require PECAM-1, whereas
IL-1
We thank Dr Peter Newman for the PITC mutant PECAM-1 construct, Drs William Muller and Robert Rothlein for the Hec7 and LR6.5 antibodies, and Dr Robert M. Streiter for anti-IL-8 serum. We thank Dr Clayton Buck for his comments and suggestions.
Submitted August 8, 2002; accepted November 18, 2002.
Prepublished online as Blood First Edition Paper, December 5, 2002; DOI 10.1182/blood-2002-08-2396.
Supported by National Institutes of Health grants HL04248 (C.D.O.) and HL49591 (S.M.A.).
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: Christopher D. O'Brien, c/o Steven M. Albelda, Department of Pulmonary/Critical Care, University of Pennsylvania, 838 BRB II/III, 421 Curie Blvd, Philadelphia, PA 19104; e-mail: christoo{at}mail.med.upenn.edu.
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© 2003 by The American Society of Hematology.
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  N. Sugimoto, T. Rui, M. Yang, S. Bharwani, O. Handa, N. Yoshida, T. Yoshikawa, and P. R. Kvietys Points of Control Exerted along the Macrophage-Endothelial Cell-Polymorphonuclear Neutrophil Axis by PECAM-1 in the Innate Immune Response of Acute Colonic Inflammation J. Immunol., August 1, 2008; 181(3): 2145 - 2154. [Abstract] [Full Text] [PDF]
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A. Verbrugge, T. de Ruiter, C. Geest, P. J. Coffer, and L. Meyaard Differential expression of leukocyte-associated Ig-like receptor-1 during neutrophil differentiation and activation J. Leukoc. Biol., April 1, 2006; 79(4): 828 - 836. [Abstract] [Full Text] [PDF]
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P. J. Newman and D. K. Newman Signal Transduction Pathways Mediated by PECAM-1: New Roles for an Old Molecule in Platelet and Vascular Cell Biology Arterioscler Thromb Vasc Biol, June 1, 2003; 23(6): 953 - 964. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
catenin, binding to the cytoplasmic domain of
PECAM-1
CD, PITC
(provided by Dr Peter Newman, The Blood Center of Southeastern
Wisconsin, Milwaukee), and AAAA (originally referred to as
KCYFLAAAA) that disrupts cell border localization. All
stably transfected cell lines, but not untransfected REN, uniformly
express PECAM-1 (> 95% cells) at levels similar to that of
RHP.
cm2 above transwell resistance, similar to
values reported (12 ±13
)
or anti-
) antibodies. (B) Transmigration
following exposure of PMN and REN or RHP monolayers to anti-
