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Prepublished online as a Blood First Edition Paper on December 5, 2002; DOI 10.1182/blood-2002-08-2396.
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Blood, 1 April 2003, Vol. 101, No. 7, pp. 2816-2825
PHAGOCYTES
PECAM-1-dependent neutrophil transmigration is independent
of monolayer PECAM-1 signaling or localization
Christopher D. O'Brien,
Poay Lim,
Jing Sun, and
Steven M. Albelda
From the Division of Pulmonary, Allergy, and Critical
Care, Department of Medicine, University of Pennsylvania School of
Medicine, Philadelphia, PA.
 |
Abstract |
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  -integrins. Although PECAM-1 modulates polymorphoneutrophil transmigration via homophilic
PECAM-1-PECAM-1 interaction, the mechanisms underlying endothelial
PECAM-1 function are unknown. Proposed mechanisms include (1) formation
of a haptotactic gradient that "guides" neutrophils to the
cell-cell border, (2) service as a "passive ligand" for neutrophil
PECAM-1, ultimately mediating activation of neutrophil integrins,
(3) regulation of endothelial calcium influx, and (4) mediation of SH2
protein association, and/or (5) catenin and non-SH2 protein
interaction. Utilizing PECAM-1-null "model" endothelial cells (REN
cells), we developed a neutrophil transmigration system to study
PECAM-1 mutations that specifically disrupt PECAM-1-dependent
signaling and/or PECAM-1 cell localization. We report that
interleukin-1 (IL-1) elicits PECAM-1-dependent
transmigration that requires homophilic PECAM-PECAM-1
engagement, but not heterophilic neutrophil PECAM-1 interactions, and
is intercellular adhesion molecule-1 dependent. Conversely,
whereas IL-8 and leukotriene-B4-mediated transmigration is PECAM-1-independent, PECAM-1 and
IL-8-dependent transmigration represent separable and additive
components of cytokine-induced transmigration. Surprisingly, neither
monolayer PECAM-1-regulated calcium signaling, cell border
localization, nor the PECAM-1 cytoplasmic domain was required for
monolayer PECAM-1 regulation of neutrophil transmigration. We conclude
that monolayer (endothelial cell) PECAM-1 functions as a
passive homophilic ligand for neutrophil PECAM-1, which after
engagement leads to neutrophil signal transduction, integrin
activation, and ultimately transmigration in a stimulus-specific manner.
(Blood. 2003;101:2816-2825)
© 2003 by The American Society of Hematology.
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Introduction |
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, that induce endothelial expression of surface proteins and
secretion of factors that ultimately activate neutrophils and promote
transmigration.2 The mechanisms regulating neutrophil
transmigration are thus stimulus- and vascular bed-specific and
involve cell adhesion proteins such as intercellular adhesion
molecule-1 (ICAM-1), the -integrins, and
others.3-6
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)-mediated (type II) neutrophil TEM but not in
chemokine-mediated (type I) TEM.15-18 However, the mechanisms by which endothelial PECAM-1 regulates TEM remain unclear.
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 2 (and 1) integrins on a variety of
leukocytes following its engagement.22,23 In neutrophils, this signaling involves PECAM-1 association with phosphatidylinositol 3-(PI3) kinase24 and PECAM-1-dependent
activation of the GTPase Rap1 via the PECAM-1 cytoplasmic
domain.25 Engagement of PECAM-1 expressed on Jurkat cells
has also been found to activate calcium signals,26 a
signaling pathway critical to leukocyte TEM.27 Thus, a
second mechanism by which endothelial PECAM-1 might regulate transmigration would be as a "passive" homophilic ligand for
neutrophil PECAM-1, activating neutrophil signal transduction
and integrins in a spatially and temporally appropriate manner.
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 and  catenin, binding to the cytoplasmic domain of
PECAM-137,38 suggest the fifth possibility, that these
interactions underlie the regulation of TEM by EC PECAM-1.
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.
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Materials and methods |
Antibodies and reagents
Anti-PECAM-1 antibodies used include monoclonal antibody
(mAb) 4G6, a murine IgG directed against extracellular loop
6,41 HEC7, a murine mAb against the extracellular loops 1 and 2 (Elias et al,42 kindly provided by Dr William
Muller, Weill Medical College of Cornell University, New York, NY), mAb
62, a murine IgG directed against extracellular loop 1,19
and Houston, a rabbit polyclonal IgG against the PECAM-1 extracellular
domain.8 Other antibodies include murine anti-MHC-1 mAb
W6-32 (American Type Culture Collection, Manassas, VA), rabbit
anti-2 microglobulin (Sigma, St Louis, MO), LR6.5 murine IgG
directed against the extracellular domain of ICAM-1 (kindly provided by
Dr Robert Rothlein, Boehringer Ingelheim, Ridgefield, CT), rabbit
anti-IL-8 serum (kindly provided by Dr Robert Streiter, University of
California, Los Angeles), nonimmune (NI) rabbit serum, and
FITC-conjugated goat antimouse (ICNCappel, Irvine, CA).
Cells and PECAM-1 mutant constructs
REN cells, a human mesothelioma cell line previously isolated in
our laboratory,43 were grown in RPMI media (Gibco BRL, Rockville, MD) supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine. The human PECAM-1
constructs utilized in this study, RHP, PECAM- CD, PITC
(provided by Dr Peter Newman, The Blood Center of Southeastern
Wisconsin, Milwaukee), and AAAA (originally referred to as
CD+KCYFLAAAA in Sun et al,39 Figure 1), were generated through sequence
overlap extension, subcloned into the pcDNA-neo vector, and
introduced using lipofectin (Gibco BRL) as described.29,39
Stable REN cell PECAM-1-transfected cell lines were established
through magnetic bead sorting and selection in G418 (0.5 mg/mL; Gibco
BRL)-supplemented media. All stably transfected cell lines, but not
untransfected REN, uniformly (> 95%) expressed high levels of
PECAM-1, demonstrated by fluorescence-activated cell sorting (FACS) and
immunoblot, at 2- to 3-fold that of HUVECs. In adenovirus transduction
experiments, REN cells were transduced 24 hours prior to experiments
with vehicle, Ad.LacZ (control adenovirus encoding the
LacZ gene), or Ad.PECAM-1 (adenovirus containing human
PECAM-1) as described.39 Adenovirus was
obtained from the University of Pennsylvania Vector Core facility, with
transduction performed at the lowest multiplicity of infection
(MOI) yielding consistent protein expression. Virus
transduction was confirmed by FACS analysis (PECAM-1) or LacZ (Gal)
assay (Promega, Madison, WI) on concurrently transduced cell
monolayers.
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| Figure 1.
PECAM-1 full-length and mutant constructs.
Schematic representation and calcium signaling and SH2 protein
interaction characteristics of the PECAM-1 constructs described in the
text. RHP indicates full-length human PECAM-1. The
extracellular domain containing 6 Ig-like loops is represented as
filled ovals 1-6, the transmembrane domain as a rectangle, and the
cytoplasmic domain as a gray rectangle, representing
cytoplasmic exons 9-16. PITC is a chimeric construct containing an
intact PECAM-1 extracellular domain fused to the nonhomologous ICAM-1
transmembrane and cytoplasmic domains. PECAM-CD is a deletion
construct lacking the PECAM-1 cytoplasmic domain. AAAA is a cytoplasmic
domain deletion construct, encoding only 9 cytoplasmic amino acid
residues, with a mutation in the membrane proximal sequence
(KCYFLRKAK 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.
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Neutrophil transmigration assay
Transmigration assays were performed as adapted from previously
reported methodologies using ECs and mesothelial
cells.5,40,44 REN cells were applied (250 000 cells/well)
to fibronectin-treated 3-µm pore, 12-mm diameter Costar transwells
(Corning, Cambridge, MA). Confluence was monitored by measurement of
transmonolayer resistance utilizing an ohmmeter adapted for 12-mm
costar wells (World Precision Instruments, Sarasota, FL). Monolayer
resistance was calculated by subtracting monolayer transwell values
from the concurrently measured resistance of transwells without cells. Confluence was indicated by development of a maximal plateau of resistance. Correlation of resistance plateaus and confluence was
confirmed through immunohistochemical staining of transwell monolayers.
Prior to transmigration assays, "luminal" cell monolayers (upper
well) were pretreated with cytokines for 24 hours or "abluminal" chemokines and chemoattractants applied (bottom well) at the time of
the assay. IL-1 (10 U/mL; Roche, Indianapolis, IN), IL-8 (5 nm; R
and D Systems, Minneapolis, MN), LTB4 (100 nm; Sigma), and tumor necrosis factor  (TNF, 100 U/mL; Roche) were
prepared on the day of assay and applied to transwells as indicated.
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-stimulated REN and RHP monolayers revealed substantial neutrophil transmigration within 1 hour that reached a maximal plateau
by 4 hours (data not shown). As previously described,5 this "maximal" plateau time was chosen to ensure that all but the
lowest MPO values would fall within the linear portion of the MPO
standard curve. Results, whether normalized to negative antibody
controls or expressed as the proportion of total PMNs (500 000),
represent averaged values from at least 2 separate experiments and at
least 3 replicates in each experiment. Data were processed with 2-way
Anova with correction for multiple comparisons utilizing Statgraphic
Plus software (Manugistics, Rockville, MD).
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).
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Results |
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.28,44,49
An 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 or IL-1, but not IL-8
(Table 1). REN cells transfected with
wild-type or mutant PECAM (not shown) had no change in PECAM expression
following exposure to IL-1 or TNF (Table 1), again similar
to ECs.21
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  cm2 above transwell resistance, similar to
values reported (12 ±13cm2) for endothelial cell
monolayers.27 In these confluent RHP monolayers grown on
transwell filters (Figure 2A), PECAM-1
demonstrated clear cell border localization, identical to that seen in
endothelial cells and RHP cells grown on glass
slides.28,39 Importantly, exposure of RHP cells to IL-1
did not alter the cell-cell distribution of PECAM-1, nor was electrical
resistance in RHP or REN monolayers affected by cytokine treatment
(data not shown), consistent with prior reports in EC
monolayers.21,27,51
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| Figure 2.
REN and PECAM-1-transfected REN cells are
phenotypically similar to ECs and support cytokine-stimulated,
ICAM-1-dependent PMN transmigration.
(A) Confluent REN and RHP monolayers plated on transwells stained with
anti-PECAM-1 antibody (mAb4G6). RHP monolayers were treated for 24 hours with IL-1 or vehicle exactly as performed in TEM experiments.
(B) PMN transmigration through confluent REN and RHP monolayers treated
for 24 hours with vehicle or 10 U/mL IL-1 (luminal). (C) PMN
transmigration though IL-1-stimulated REN and RHP monolayers.
Monolayers and PMNs were exposed to either isotype-matched anti-MHC-1
or anti-ICAM-1 mAbs (100 µg/mL) 20 minutes prior to and during
transmigration. Identical results were obtained using BSA (100 µg/mL)
for nonblocking conditions (not shown). Transmigration rates are
expressed as the proportion of PMNs migrating through the transwell
filter compared with the total number of PMNs added (500 000). In
these representative experiments, data represent the mean ± SEM from a
minimum of 3 transwells for each condition. (*Significantly different
from "unblocked" controls, P < .05.)
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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 (type
II transmigration).2 As shown in Figure 2B, in 4-hour
neutrophil (PMN) transmigration experiments utilizing 500 000
PMNs/well, fewer than 5% of total PMNs transmigrated through
unstimulated REN and RHP monolayers. However, following pretreatment
with IL-1, REN and RHP monolayers supported a dramatic increase in
PMN transmigration (20%-40% of PMNs transmigrating). Transmigration
through IL-1-stimulated RHP monolayers was about double that seen
in the IL-1-stimulated REN monolayers. In addition, when the
chemoattractants IL-8 or LTB4 were placed in the lower chamber of the transwell, a marked increase in transmigration also
occurred, though at identical levels in both cell types (Figure 4).
As seen in Figure 2C, we found that proportionally, more than two
thirds of IL-1-stimulated transmigration in both REN and RHP
monolayers was blocked by anti-ICAM-1 antibodies compared with wells
treated with the isotype-matched anti-MHC-1-negative control mAb or
BSA (not shown). These findings suggest that in both cell lines,
similar to most EC beds, the majority of transmigration is ICAM-1
dependent.4,52
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-stimulated transmigration, whereas the (small) residual ICAM-1-independent portion of transmigration observed in
both REN and RHP monolayers does not appear to be significantly
impacted by the presence (or absence) of PECAM-1.
Role of PECAM-1 in IL-1-mediated type II neutrophil
transmigration
In order to confirm the role of PECAM-1 in neutrophil diapedesis,
we first compared the transmigration rate of human neutrophils across
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.
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| Figure 3.
IL-1-stimulated PMN transmigration is composed of
PECAM-1-dependent and -independent components.
Cell monolayers were treated for 24 hours with 10 U/mL IL-1
(luminal). Transmigration rates are expressed as the proportion of
transmigrating PMNs compared with "unblocked" controls (set at
1.0). Data represent the mean ± SEM from a minimum of 3 transwells for
each condition from at least 3 separate experiments unless otherwise
indicated. (*Significantly different from "unblocked" controls,
P < .05.) (A) Transmigration following exposure of
neutrophils and REN or RHP monolayers to anti-PECAM-1 (Houston,  )
or anti-2 microglobulin ( ) antibodies. (B) Transmigration
following exposure of PMN and REN or RHP monolayers to anti-2
microglobulin (reference set at 1.0), anti-MHC-1, anti-Gal
antibodies, BSA, or anti-PECAM-1 (Houston) antibodies (all 100 µg/mL). (C) RHP cells. Luminal BSA ("unblocked" reference
control) or anti-PECAM-1 antibodies Houston, mAb62, Hec7, or 4G6 (100 µg/mL) were added as indicated. Data represent the mean ± SEM from a
minimum of 3 transwells for each condition from at least 2 separate
experiments. (D) Confluent REN monolayers transduced with Ad.PECAM-1 or
Ad.LacZ (PECAM null) and subsequently treated for 24 hours with 10 U/mL
IL-1 (luminal). PMNs and tranduced REN monolayers were exposed to
anti-PECAM-1 (Houston, ) or anti-MHC-1 () antibodies as
indicated. Values are normalized to LacZ transfected (PECAM-1 null),
"unblocked" (anti-MHC-1 antibody-treated), negative control.
(*Significantly different from Ad.LacZ negative controls,
P < .05; **significantly different from "unblocked"
Ad.PECAM-1, P < .05.)
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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-2 microglobulin-negative control standard. In
IL-1-stimulated RHP cells, there was no significant difference in
TM among control conditions (including BSA alone, an irrelevant rabbit
polyclonal anti-Gal antibody, an anti-MHC-1 monoclonal antibody,
and the polyclonal anti-2 microglubulin antibody), while
anti-PECAM-1 blocking antibody (Houston) manifested significant
blockade compared with all controls (38%-50%). In IL-1-stimulated
REN cells, there was no significant difference in transmigration among
any conditions (Figure 3B), confirming our prior findings (Figure 3A).
Thus, although we used intact antibodies, there was no evidence of Fc receptor activation of neutrophils by the rabbit polyclonal or mouse
monoclonal antibodies. Similarly, we found no difference in MPO content
between neutrophils exposed to media, BSA, or any control or
anti-PECAM-1 antibodies (data not shown). Having previously demonstrated that neither anti-MHC-1 mAb nor BSA elicits calcium signaling activity in ECs or RHP28,29 and given their
equivalence to polyclonal anti-2 microglobulin as negative controls,
we utilized these well-characterized control reagents in our subsequent
transmigration experiments.
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-treated REN cells compared with BSA, anti-MHC-1, or
anti-Gal antibody controls (not shown).
In order to assess the contribution of PECAM-1 expression to
IL-1-stimulated transmigration and to rule out artifacts resulting from phenotypic variations between permanently transfected cell lines,
we performed TM experiments on IL-1-stimulated REN monolayers transduced with adenovirus containing human PECAM-1 (Ad.PECAM-1) or a
control adenovirus encoding the LacZ gene (Ad.LacZ). This approach resulted in PECAM-1 expression comparable with that of RHP
cells in more than 90% to 95% of cells (not shown). Figure 3D shows
that in unblocked Ad.PECAM-1-transduced REN cells, we observed an
average 75% increase in PMN transmigration compared with unblocked
Ad.LacZ (PECAM-1 null) control monolayers
(P < .05). This increase was completely blocked in
Ad.PECAM-1-transduced REN monolayers treated with anti-PECAM-1
antibodies, whereas anti-PECAM-1 antibody treatment of
Ad.LacZ-transduced monolayers did not diminish transmigration. These
data confirm the following: (1) PECAM-1-dependent transmigration
requires monolayer expression of PECAM-1; (2) neutrophil PECAM-1
heterophilic interactions are not required; and (3) IL-1-stimulated transmigration consists of both a PECAM-1-dependent component (antibody-blockable) and a PECAM-1-independent component
(which is supported by Ad.LacZ-transduced REN or antibody-blocked
Ad.PECAM-1-transduced REN monolayers).
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 increased PMN
binding to REN and RHP monolayers similarly when compared with
untreated monolayers. However, this effect was not blocked by addition
of anti-PECAM-1 or anti-MHC-1 antibodies (not shown). Additionally,
at the end of the 4-hour TEM assays, fewer than 7% of total PMNs were
"trapped" in the transwell inserts (or firmly adherent to the
luminal or abluminal side) with no significant difference in cell
retention found between REN and RHP cells or between antibody
conditions (not shown). These results indicate that the differences in
PMN transmigration we have interpreted as the "PECAM-1-dependent"
component of TEM are not simply an artifact of neutrophil sequestration.
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.
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| Figure 4.
PECAM-1-dependent transmigration is chemokine/cytokine
specific.
Confluent RHP and REN monolayers treated with 5 nm IL-8 (abluminal) or
100 nm LTB4 (abluminal) at the start of the TM assay or
with 10 U/mL IL-1 (luminal) for 24 hours as indicated. Monolayers
and neutrophils were treated with luminal anti-PECAM-1 (Houston) or
anti-MHC-1 antibodies (100 µg/mL) as indicated. Similar results were
obtained using BSA instead of anti-MHC-1 antibody (not shown).
Transmigration rates are expressed as the proportion of PMNs migrating
through the transwell filter compared with the total number of PMNs
added at the start of the experiment. Data represent the mean ± SEM
from a minimum of 3 transwells for each condition from 2 separate
experiments. (*Significantly different from all other columns,
P < .05.)
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Relationship of IL-8 (type I) transmigration and PECAM-1 in
IL-1-mediated (type II) transmigration
It is well known that IL-8 levels produced by 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).
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| Figure 5.
IL-1-stimulated TEM consists of PECAM-1 and
IL-8-dependent components that are separable and additive.
(A) Confluent RHP monolayers were treated with 5 nm IL-8 (abluminal)
and either 1:200 negative control rabbit serum (NC) or anti-IL-8 serum
was added to the abluminal (bottom) chamber at the start of the TM
assay. PMNs were allowed to transmigrate for 4 hours. In this
representative experiment, data represent the mean ± SEM from a
minimum of 3 transwells for each condition. (B) Confluent RHP
monolayers were treated for 24 hours with 10 U/mL IL-1 (luminal),
washed, and 500 000 PMNs/well were applied to the luminal chamber.
Anti-IL-8 serum (1:200) or negative control NI rabbit serum was added
to either the luminal (top) or abluminal (bottom) chamber in
conjunction with (luminal) anti-PECAM-1 (Houston) or anti-MHC-1
antibodies (100 µg/mL) as indicated (PMN aliquots were treated
identically to luminal conditions 20 minutes prior to addition to
wells). PMNs were allowed to transmigrate for 4 hours. Transmigration
rates are expressed as the proportion of PMNs migrating through the
transwell filter compared with the total number of PMNs added at the
start of the experiment. Column 1, "unblocked" TM (anti-MHC-1) in
the setting of abluminal NI serum; Column 2, "unblocked" TM
(anti-MHC-1) in the setting of abluminal anti-IL-8 serum; Column 3, "blocked" TM (Houston) in the setting of abluminal NI serum; Column
4, "blocked" TM (Houston) in the setting of abluminal anti-IL-8
serum; Column 5, "unblocked" TM (anti-MHC-1) in the setting of
luminal NI serum; Column 6, "unblocked" TM (anti-MHC-1) in the
setting of luminal anti-IL-8 serum. Data represent the mean ± SEM
from a minimum of 3 transwells for each condition from 3 separate
experiments. (*Columns 2, 3, and 4 are significantly different
[P < .05] from column 1. **Column 4 is significantly
different from columns 2 and 3 [P < .05].)
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In contrast, in IL-1-stimulated RHP monolayers incubated with
anti-MHC-1 antibody and abluminal anti-IL-8 serum (column 2), PMN
transmigration was inhibited by 40% compared with abluminal NI control
(column 1). These findings are consistent with previous reports that
IL-1 treatment of EC monolayers leads to secretion of IL-8 that may
play a role as a chemoattractant in PMN transmigration. Utilizing
enzyme-linked immunosorbent assay (ELISA) techniques, we
confirmed that IL-1-pretreated REN and RHP monolayers secrete IL-8,
whereas untreated monolayers do not (data not shown).
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-mediated PMN transmigration. In anti-PECAM-1-treated (Houston) monolayers to which abluminal anti-IL-8 serum was also added
(column 4), an 86% decrease in PMN migration was observed (compared
with column 1). These findings suggest that IL-8 (type I) and
PECAM-1-regulated transmigration are independent and additive components of IL-1-mediated (type II) PMN transmigration.
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-stimulated PITC and RHP monolayers were conducted, treatment
with anti-PECAM-1 antibodies (Houston) yielded a more than 80%
decrease in transmigration compared with anti-MHC-1 negative control.
Similar findings were observed with BSA negative control (not shown).
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| Figure 6.
PECAM-1-dependent
transmigration does not require the PECAM-1 cytoplasmic domain or cell
border localization.
(A) IL-1-treated RHP and PITC monolayers exposed to anti-PECAM-1
(Houston) or anti-MHC-1 antibodies. (B) IL-1-treated RHP and
PECAM-CD monolayers exposed to anti-PECAM-1 (Houston) antibodies or
BSA (100 µg/mL). Similar results were obtained with anti-MHC-1
antibodies instead of BSA (not shown). (C) IL-1-treated RHP and
AAAA monolayers exposed to anti-PECAM-1 (Houston) antibodies or
anti-MHC-1 antibodies. Similar results were obtained with BSA instead
of anti-MHC-1 antibodies (not shown). Transmigration rates are
expressed as the normalized proportion of migrating PMNs compared with
"unblocked" TM (set at 1.0) for each cell line. Data represent the
mean ± SEM from a minimum of 3 transwells for each condition from 3 separate experiments. (*Significantly different from "unblocked"
negative control, P < .05.)
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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-CD, lacks the entire cytoplasmic domain and is known to
disrupt cell border localization, as well as PECAM-1-dependent calcium
and tyrosine signaling events,30,39 but can serve as a
ligand for homophilic PECAM-1 binding (not shown). However, because
ICAM-1 expression in PECAM-CD was, for some reason, significantly lower than in RHP cells (Table 2), we
also utilized a second cytoplasmic deletion construct, termed AAAA,
encoding only 9 cytoplasmic amino acid residues with a mutation in the
membrane proximal charged stop-transfer sequence
(KCYFLRKAKKCYFLAAAA) that is also known to disrupt cell border
localization.39 Notably, ICAM-1 expression before and
after IL-1 treatment in REN-AAAA cells was similar to REN, RHP, and
PITC cells (Tables 1-2). In TM experiments comparing PECAM-CD and
AAAA to RHP cell monolayers, there was an approximately 50% to 60%
inhibition of PMN transmigration with anti-PECAM-1 antibodies in both
cell lines (Figure 6B-C) compared with nonblocking control, similar to
that seen in RHP controls. This indicates that the PECAM-1-dependent
component of IL-1-mediated TEM does not require PECAM-1 cell border
localization or signaling and is independent of absolute ICAM-1
expression levels.
| |
Discussion |
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]-mediated) and type I (chemokine [IL-8]- and
chemoattractant [LTB4]-mediated) transmigration. In cell
monolayers treated with IL-1, PMN transmigration on
PECAM-1-expressing REN cells was enhanced compared with untransfected
REN cells and was partially blocked by anti-PECAM-1 antibodies against
PECAM-1 extracellular loops 1 or 2, while no anti-PECAM-1 antibodies
disrupted PMN TM through PECAM-1-"null" REN. These data
demonstrate the presence of a PECAM-1-dependent component to
cytokine-induced transmigration that requires homophilic interaction
between monolayer and PMN PECAM-1 and is independent of heterophilic
interactions between neutrophil PECAM-1 and EC non-PECAM-1 ligands.
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)-mediated TM is composed of both PECAM-1-dependent and PECAM-1- independent (IL-8-dependent)
components that are separable and additive, further refining previous
observations that cytokine-mediated TEM involves expression of
chemokines such as IL-8.45,46,54 Furthermore, these
data suggest a mechanism for the PECAM-dependent and PECAM-independent
components of transmigration after prolonged IL-1 exposure. Unlike
"pure" type I transmigration,2 in which
large amounts of direct neutrophil activators are present and
neutrophils do not require a "prestimulated" monolayer (Figure 4), transmigration after IL-1 stimulation of the monolayer appears to
involve, in part, neutrophil activation caused by secreted IL-8 (at low
concentrations), while another component of transmigration requires
neutrophil activation through contact-dependent PECAM-1 interactions
with the "prestimulated" monolayer.
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-de |
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Abstract
Introduction