|
|
Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 3685-3693
Monocyte Arrest and Transmigration on Inflamed Endothelium in Shear
Flow Is Inhibited by Adenovirus-Mediated Gene Transfer of I B-
By
Kim S.C. Weber,
Georg Draude,
Wolfgang Erl,
Rainer de Martin, and
Christian Weber
From the Institut für Prophylaxe und Epidemiologie der
Kreislaufkrankheiten, Ludwig-Maximilians Universität,
München, Germany; the Karolinska Hospital, Centre of Molecular
Medicine L8:03, Stockholm, Sweden; and the Vienna International
Research Center, Department of Vascular Biology, Vienna, Austria.
 |
ABSTRACT |
Mobilization of nuclear factor- B (NF- B) activates
transcription of genes encoding endothelial adhesion molecules and
chemokines that contribute to monocyte infiltration critical in
atherogenesis. Inhibition of NF- B has been achieved by
pharmacological and genetic approaches; however, monocyte interactions
with activated endothelium in shear flow following gene transfer of the
NF- B inhibitor I B- have not been studied. We found that
overexpression of I B- in endothelial cells using a recombinant
adenovirus prevented tumor necrosis factor- (TNF- )-induced
degradation of I B- and suppressed the upregulation of vascular
cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1
(ICAM-1), and E-selectin mRNA and surface protein expression and the
upregulation of transcripts for the chemokines monocyte chemoattractant
protein 1 (MCP-1) and growth-related activity- (GRO- ) by
TNF- . This was associated with a reduction in
endothelial MCP-1 secretion and GRO- immobilization. Adhesion assays
under physiological shear flow conditions showed that firm arrest,
spreading, and transmigration of monocytes on TNF- -activated
endothelium was markedly inhibited by I B- overexpression. Inhibition with monoclonal antibodies and peptide antagonists inferred
that this was due to reduced expression of Ig integrin ligand as well
as of chemokines specifically involved in these events. In contrast,
rolling of monocytes was increased by I B- transfer and was partly
mediated by P-selectin; however, it appeared to be unaffected by the
inhibition of E-selectin induction. Thus, our data provide
novel evidence that selective modulation of NF- B by adenoviral
transfer of I B- impairs the expression of multiple endothelial
gene products required for subsequent monocyte arrest and emigration in
shear flow and thus for monocyte infiltration in atherosclerotic plaques.
© 1999 by The American Society of Hematology.
 |
INTRODUCTION |
MONOCYTE ADHESION and emigration involves
sequential and overlapping interactions of signal molecules and is
fundamental in the initial pathogenesis and progression of
atherosclerosis.1-3 Whereas monocyte rolling on endothelium
is mediated by selectins, activation of 1 and 2 integrins that
bind to their endothelial ligands, vascular cell adhesion molecule-1
(VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), respectively,
is critical for firm adhesion and transmigration.2,3 The
expression of VCAM-1 and ICAM-1 has been demonstrated in coronary
atherosclerotic lesions, particularly in areas of neovascularization,
and may contribute to leukocyte recruitment.4,5 Moreover,
the CC chemokine monocyte chemoattractant protein 1 (MCP-1), which can
trigger chemotaxis and extravasation of monocytes via its receptor
CCR2, has been detected in atherosclerotic plaques.6-9 In
addition, the induction and immobilization of the CXC chemokine
growth-related activity- (GRO- ) can induce monocyte adhesion to
endothelium stimulated by minimally modified low-density lipoprotein
(LDL), implying a role in monocyte recruitment during
atherogenesis.10 Inflammatory cytokines, such as tumor
necrosis factor- (TNF- ), upregulate the endothelial adhesion
molecules ICAM-1, VCAM-1, and E-selectin and the production of
chemokines, eg, MCP-1, at the level of gene transcription involving the
binding of nuclear factor- B (NF- B) to motifs in the promoter
regions.11-14 Activation and nuclear translocation of
NF- B occurs in endothelial cells and requires the phosphorylation of
its inhibitor I B- , which is degraded by a proteasome-dependent
pathway.15-18
Inhibition of NF- B by increasing levels of I B- in endothelial
cells has been suggested to be of potential use in suppressing the
inflammatory response.19-22 Adenovirus-mediated gene
transfer may be a potentially specific and effective method of gene
delivery into local areas of inflammation, such as atherosclerotic
plaques or after balloon angioplasty.23-25 We used an
adenoviral vector encoding I B- to inhibit NF- B activation and
found that gene transfer of I B- in endothelial cells impaired the
TNF- -induced upregulation of adhesion molecule and chemokine
expression. This was associated with a reduction in firm adhesion and
transmigration but not in rolling of monocytes on activated endothelium
in shear flow. These data extend the understanding of the mechanisms of action exerted by adenovirus-mediated inhibition of NF- B as a potential therapy in the prevention of atherogenesis and its consequences.
 |
MATERIALS AND METHODS |
Cell culture and reagents.
Human umbilical vein endothelial cells (HUVEC) were used at passages 2 to 4 and grown in low serum PromoCell medium,26 and Mono
Mac 6 cells (from Dr H.W.L. Ziegler-Heitbrock, Institut für Immunologie, Munich, Germany) were maintained as
described.27,28 Monocytes were isolated from healthy human
donors by NycoPrep density gradient centrifugation (Nycomed,
Norway) and separated from platelets by multiple low
gravity washes, as described.29 This protocol
resulted in a purity of greater than 85% monocytes and a minimal
contamination with platelets (<5%), as assessed by light scatter,
staining for CD14 and P-selectin, and subsequent flow cytometric
analysis that showed an overwhelming majority of P-selectin-negative
monocytes (data not shown).
The ICAM-1 monoclonal antibody (MoAb) RR1/130 was from Dr
R. Rothlein (Boehringer Ingelheim, CT). The 2 MoAb
TS1/18 was from Dr L.B. Klickstein (Brigham & Women's Hospital,
Boston, MA),31 the 4 MoAb HP1/2 from Dr R. Lobb (Biogen, Cambridge, MA),32 and the
blocking G1 and nonblocking S12 MoAbs to P-selectin from Dr R. McEver
(University of Oklahoma).33,34 The peptide
analogues 8-73 GRO- and 9-76 MCP-135,36 were kind gifts
from Dr I. Clark-Lewis (University of British Columbia, Vancouver,
Canada). MoAbs to VCAM-1, E-selectin, MCP-1, or GRO-
and isotype controls were from Canon or R&D Systems (Wiesbaden,
Germany) and reagents were from Sigma Chemical Co (Deisenhofen,
Germany), unless otherwise stated.
Overexpression of adenovirally encoded
I B- in endothelial cells.
Construction of the adenoviral vector encoding for I B-
(rAd.I B- ) and infection of endothelial cells were performed as previously described.20 Briefly, confluent HUVEC were
washed with phosphate-buffered saline (PBS) and incubated with the
adenovirus (multiplicity of infection [moi] of 100) in
PBS for 30 minutes at 37°C, washed, and then cultured for 48 hours.
Infection with a control adenoviral vector37 encoding green
fluorescence protein (rAd.GFP) was performed at an equivalent moi of
100, and effective transduction was confirmed by analyzing GFP
expression by flow cytometry, as described.37 Unless
otherwise stated, cells were stimulated with TNF- (100 U/mL) for 4 hours. Overexpression of I B- may induce apoptosis in
TNF- -activated endothelial cells by suppressing induction of
inhibitor of apoptosis proteins38; however, cell viability
was determined to be greater than 95% under all conditions. For
Western blot, cells left untreated or activated with TNF- for 1 hour
were lysed in sample buffer containing protease inhibitors and lysates
were separated by 12.5% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). Proteins were transferred to nitrocellulose
membranes and reacted with an MoAb to I B- (Santa Cruz
Biotechnology, Heidelberg, Germany). Blots were developed
with chemiluminescence (ECL; Amersham, Braunschweig, Germany).
Reverse transcription-polymerase chain reaction (RT-PCR).
Total RNA was isolated by phenol/chloroform/isoamylalcohol extraction
and cDNA was reverse transcribed from 2 µg RNA. PCR was performed and
PCR products were analyzed by agarose gel electrophoresis and
quantitated by high-performance liquid chromatography
(HPLC) as described.26,39 Primer sequences
were TTGCAGACCCTGCAGGGAAT (GRO- , sense), TGGATTTGTCACTGTTCAGC
(GRO- , antisense), GTCTCTGCAACGCTTCTGTGCC (MCP-1, sense), AGTCG
TGTGTCTTGGGTTGTGG (MCP-1, antisense), AGTAATAGTCCTCCTCATCATG (E-selectin, sense), and ACCATCTCAAGTGAAGAAAGAG (E-selectin, antisense) and as published for ICAM-1, VCAM-1, and -actin.39
Flow cytometry and quantification of MCP-1 protein.
Confluent HUVEC were trypsinized, washed, and reacted with saturating
concentrations of MoAb for 30 minutes on ice, washed, and stained with
fluorescein isothiocyanate (FITC)-or phycoerythrin (PE)-conjugated goat
antimouse IgG (Boehringer Mannheim, Mannheim, Germany),
washed, and analyzed in a FACScan (Becton Dickinson, Heidelberg,
Germany).26,39 The concentration of MCP-1
protein present in the HUVEC supernatants was determined using a
sandwich enzyme-linked immunosorbent assay (ELISA; R&D Systems)
performed according to the manufacturer's protocols.
Monocyte adhesion and transmigration on endothelium in shear flow.
Laminar flow assays were performed as previously
described.3,29,40 HUVEC were grown to confluence in 35-mm
petri dishes that were assembled as the lower wall in a parallel wall
flow chamber and mounted on the stage of an Olympus IMT-2 inverted microscope (Olympus Optical, Hamburg, Germany) with
20× and 40× phase contrast objectives. Monocytes (0.5 × 106/mL) or Mono Mac 6 cells (106/mL)
suspended in Hanks' buffered salt solution containing 10 mmol/L HEPES,
pH 7.4, 0.5% human serum albumin, and 1 mmol/L Mg2+, 1 mmol/L Ca2+ added shortly before the assay) were kept in a
heating block at 37°C during assays and were perfused into the flow
chamber at a rate of 1.5 dyn/cm2 for 5 minutes. The number
of firmly adherent cells after 5 minutes was quantitated in multiple
fields (at least 5 per experiment) by analysis of images recorded with
a long integration JVC 3CCD video camera and a JVC SR L 900 E video
recorder (JVC, Japan), and expressed as cells per
square millimeter. The type of adhesion analyzed was restricted to
primary, ie, direct interactions of monocytes with endothelium.
Consistent with findings using a similar flow chamber,3
secondary attachment to already adherent monocytes that is mediated by
L-selectin and results in the formation of linear
strings41-43 occurred only sporadically at 1.5 dyn/cm2, accounting for a very small component of total
interactions. The extent of secondary tethers may vary due to
differences in the dimension, profile, and other design characteristics
of the flow chambers used. The number of cells remaining bound,
undergoing shape change, or transmigrating after 5-minute intervals was
determined in high power fields, as described,2 and
expressed as the percentage of cells that had firmly adhered. As an
inverse measure of firm arrest, the number of cells rolling at reduced
velocity on endothelium was determined within the last 30 seconds of
the 5-minute intervals and was expressed as the percentage of all cells
interacting with HUVEC in the field. Pretreatment with MoAbs was
performed at 10 µg/mL. Data are expressed as the mean ± SD.
Statistics.
Statistical significance was determined by analysis of variance, and
differences with P < .05 were considered to be significant.
 |
RESULTS |
Degradation and adenoviral overexpression of
I B- in activated HUVEC.
To study the effects of I B- overexpression, HUVEC were infected
with a recombinant adenovirus encoding I B- .20 After 48 hours in culture, cells were challenged with TNF- (100 U/mL) for
1 hour and the expression of I B- was determined by Western blot.
In unstimulated cells, I B- was observed as a band of 37 kD
(Fig 1, lane 1). Treatment of cells with
TNF- resulted in a marked decrease in detectable I B- compared
with untreated cells, consistent with the degradation of I B- (Fig
1, lane 2).17,18 Cells infected with rAd.I B-
expressed substantially more I B- than untreated cells, indicating
the high efficiency of infection and protein expression (Fig 1, lane
3). Stimulation with TNF- after rAd.I B- infection decreased
the expression of I B- , which, however, remained higher than in
uninfected cells (Fig 1, lane 4). This was confirmed by densitometrical
analysis (data not shown). These data suggest that overexpression of
I B- prevents a substantial I B- degradation and NF- B
mobilization induced by TNF- .

View larger version (36K):
[in this window]
[in a new window]
| Fig 1.
Expression of I B- in HUVEC. Confluent HUVEC were
left untreated (lanes 1 and 2), infected with rAd.I B- (moi 100;
lanes 3 and 4), and/or stimulated with TNF- for 1 hour (100 U/mL;
lanes 2 and 4). Cell lysates were separated by 12.5% SDS-PAGE and
Western blot was performed with MoAb to I B- . Shown is a
representative experiment.
|
|
I B- inhibits mRNA transcription of
endothelial signal molecules.
Activation of NF- B is involved in upregulating the mRNA
transcription of endothelial adhesion molecules and chemokines
important in monocyte adhesion and
transmigration.11-14,44-46 We used RT-PCR analysis with
HPLC quantification to study the effect of I B- on the induction
of mRNA transcripts. Treatment of HUVEC with TNF- (100 U/mL) for 4 hours regulated ICAM-1, VCAM-1, and E-selectin mRNA expression
(Fig 2A), whereas overexpression of
I B- resulted in a reduction in mRNA levels after TNF-
stimulation (Fig 2A). Similarly, TNF- increased the mRNA
transcription of GRO- and MCP-1, whereas infection with
rAd.I B- inhibited these effects (Fig 2B).

View larger version (24K):
[in this window]
[in a new window]
| Fig 2.
Adenoviral I B- transfer inhibits TNF- -induced
endothelial adhesion molecule and chemokine mRNA expression. Cells were
left untreated, infected with rAd.I B- , and/or stimulated with
TNF- (100 U/mL) for 4 hours. RT-PCR was performed using specific
primers for ICAM-1, VCAM-1, and E-selectin (A); for MCP-1 and GRO-
(B); and for -actin. Quantification was perfomed by HPLC analysis,
and mRNA expression was reported as the percentage of -actin serving
as an internal standard. Data are the mean ± SD of three separate
experiments.
|
|
Inhibition of TNF- -induced adhesion molecule
expression by I B- .
We determined whether rAd.I B- infection impaired the surface
expression of endothelial adhesion molecules by flow cytometric analysis. Untreated HUVEC expressed moderate levels of ICAM-1 and low
levels of VCAM-1 and E-selectin (Fig 3A).
Stimulation with TNF- (100 U/mL for 4 hours) markedly increased the
expression of ICAM-1, VCAM-1, and E-selectin (Fig 3B through D).
Infection with rAd.I B- almost completely inhibited the
upregulation of adhesion molecules induced by TNF- (Fig 3B through
D). Thus, overexpression of I B- prevented the upregulation of
endothelial adhesion molecule surface expression.

View larger version (32K):
[in this window]
[in a new window]
| Fig 3.
Flow cytometric analysis of endothelial adhesion molecule
after I B- overexpression. (A) Untreated HUVEC were stained with
MoAbs to ICAM-1 (stippled line), VCAM-1 (dashed line), E-selectin
(dotted line), and isotype control (solid line). (B through D) Cells
stimulated with TNF- (100 U/mL) for 4 hours were stained with MoAbs
(solid line) to ICAM-1 (B), VCAM-1 (C), E-selectin (D), or isotype
control (B through D, dotted lines). Cells infected with rAd.I B-
and stimulated with TNF- (100 U/mL for 4 hours) were stained with
MoAbs (stippled line) to ICAM-1 (B), VCAM-1 (C), E-selectin (D), or
isotype control (B through D, dashed lines). Shown is a representative
experiment.
|
|
To control for effects of adenoviral infection itself, we infected
HUVEC with the control vector rAd.GFP37 encoding GFP at an
equivalent moi of 100 under identical conditions. Flow cytometric analysis showed a marked GFP expression in greater than 90% of HUVEC
after 48 hours, confirming a high transduction efficiency (Fig 4A). Notably, infection of HUVEC with
rAd.GFP under these conditions did not affect the surface expression of
ICAM-1 in unstimulated HUVEC (Fig 4B) or in HUVEC stimulated with
TNF- (100 U/mL) for 4 hours (Fig 4C).

View larger version (16K):
[in this window]
[in a new window]
| Fig 4.
Flow cytometric analysis of endothelial adhesion molecule
after infection with control vector rAd.GFP encoding GFP. (A) The
expression of GFP in HUVEC was analyzed by flow cytometry after
infection with rAd.GFP (solid line) or no treatment (dotted line) after
48 hours. (B and C) HUVEC infected with or without GFP control
adenovirus were stimulated without (B) or with (C) TNF- (100 U/mL)
for 4 hours and were stained with MoAb to ICAM-1 (solid or stippled
line) or isotype control (dotted or dashed line). Shown are
representative histograms.
|
|
Effect of I B- on endothelial
secretion of chemokines.
It has been shown that MCP-1 is secreted by endothelium as a soluble
protein, whereas GRO- can be immobilized on the endothelial surface.10,47 An ELISA of HUVEC supernatants showed that
stimulation with TNF- for 4 hours induced an increase in secretion
of MCP-1 protein that was completely prevented by overexpression of
I B- in HUVEC (Fig 5A). To determine
the effects of rAd.I B- on immobilized GRO- , we measured the
amount of surface-associated protein by flow cytometric analysis. We
found that TNF- stimulation increased the amount of GRO-
associated with the endothelial surface that was inhibited by
overexpression of I B- (Fig 5B).

View larger version (27K):
[in this window]
[in a new window]
| Fig 5.
Endothelial production of chemokines. HUVEC were left
untreated, were infected with rAd.I B- and/or stimulated with
TNF- (100 U/mL) for 4 hours. (A) Quantification of soluble MCP-1 was
performed by ELISA. Data are the mean ± SD of three separate
experiments performed in duplicate. (B) Flow cytometric analysis was
performed to quantitate the amount of surface-associated GRO- . Data
are the mean ± SD of three separate experiments.
|
|
Effect of I B- on monocyte
interactions with endothelium in shear flow.
Firm adhesion of monocytes to endothelium under shear flow involves the
4 and 2 integrins, known to interact with VCAM-1 and ICAM-1,
respectively.2,3 Moreover, chemokines such as IP10, Mig,
and eotaxin have been implicated in the arrest of T effector cells and
eosinophils in shear flow, respectively.40,48 Recently, we
have shown that surface-bound GRO- is involved in monocyte arrest,
whereas soluble MCP-1 mediates transmigration of monocytes on activated
endothelium in shear flow.49 Hence, we studied the effects
of I B- overexpression on monocyte adhesion to activated
endothelium under physiological flow conditions. After 48 hours in
culture, HUVEC infected with rAd.I B- or left untreated were
stimulated with TNF- (100 U/mL) for 4 hours and inserted as the
bottom wall of a parallel flow chamber. Isolated human blood monocytes
or monocytic Mono Mac 6 cells, which express a similar array of
adhesion molecules and chemokine receptors,50 were perfused
through the chamber at a constant shear rate of 1.5 dyn/cm2. After a 5-minute period of accumulation, the
number of Mono Mac 6 cells firmly attached to uninfected HUVEC treated
with TNF- was determined to be 198 ± 46 cells/mm2
(Fig 6A). By comparison, overexpression of
I B- in HUVEC before TNF- stimulation resulted in a significant
inhibition in the number of cells firmly arrested after 5 minutes (59 ± 22 cells/mm2), whereas infection with the control
vector rAd.GFP had no effect and thus did not confound cell arrest (Fig
6A). Because leukocytes can bind to adherent leukocytes via
L-selectin,41-43 only direct interactions of monocytes with
endothelium were included in this analysis. As described,2
a percentage of firmly attached cells was observed to undergo shape
change or spreading and migrate towards interendothelial junctions and
transmigrate. On uninfected HUVEC treated with TNF- , 25.0% ± 5.7% of the firmly attached cells were found to undergo a shape
change, spreading, or transmigration (Fig 6B). Infection of HUVEC with
rAd.I B- but not with control GFP adenovirus resulted in a clear
reduction in the fraction of cells spreading or transmigrating (Fig
6B).

View larger version (36K):
[in this window]
[in a new window]
| Fig 6.
Effects of adenoviral-mediated I B- expression on
firm adhesion, transmigration, and rolling of monocytic cells on
endothelial cells under physiological flow. HUVEC grown in 35-mm Petri
dishes were left untreated ( ), were infected with rAd.I B-
( ), or were infected with the control vector rAd.GFP encoding GFP
( ). All cells were stimulated with TNF- (100 U/mL) for 4 hours.
Mono Mac 6 cells were perfused at a constant flow rate of 1.5 dyn/cm2. (A) The firm, shear-resistant attachment of
monocytes to HUVEC was determined by counting the number of cells
firmly attached per field after a 5-minute period. (B) Cells undergoing
shape change and spreading or transmigration were counted after 5 minutes and expressed as the percentage of cells initially attached.
Data are the mean ± SD of three independent experiments. *P < .05 versus uninfected control.
|
|
Analysis of experiments performed with isolated human blood monocytes
showed that 110 ± 26 cells/mm2 had firmly attached
after a 5-minute period on uninfected and TNF- -treated HUVEC at a
shear rate of 1.5 dyn/cm2 (Fig
7A). Of these firmly attached cells, 27.5% ± 4.9% appeared to
spread or to transmigrate between endothelial cells (Fig 7B). Consistent with previous results,2,3 a combination of 4 and 2 MoAbs markedly reduced firm arrest, as well as spreading and
transmigration of monocytes (Fig 7A and B). Similarly, infection of
HUVEC with rAd.I B- significantly inhibited the shear-resistant adhesion of monocytes to TNF- -activated endothelium as compared with uninfected controls (Fig 7A). Subsequently, the percentage of
attached cells that underwent spreading or transmigration was lower on
HUVEC overexpressing I B- (Fig 7B). Consistent with recent
findings,49 the inhibition with specific peptide
antagonists showed that GRO- is involved in monocyte arrest (Fig
7A), whereas MCP-1 contributes to monocyte transmigration on activated
endothelium in shear flow (Fig 7B). Control peptides had no effect
(data not shown). Preincubation of monocytes with a combination of
blocking 4 and 2 MoAb in addition to infection of HUVEC with
rAd.I B- resulted in a slightly although not significantly greater
inhibition of monocyte arrest and of spreading/transmigration as
compared with infection with rAd.I B- or MoAb treatment alone (Fig
7A and B). Taken together, these data infer that, beyond effects on
endothelial Ig integrin ligands, the reduced chemokine expression in
rAd.I B- -infected HUVEC is at least in part responsible
for the inhibition of arrest and transmigration.

View larger version (21K):
[in this window]
[in a new window]
| Fig 7.
Effects of adenoviral-mediated I B- expression on
firm arrest, transmigration, and rolling of isolated blood monocytes on
endothelial cells under physiological flow conditions. HUVEC were left
untreated or were infected with rAd.I B- and were stimulated with
TNF- (100 U/mL) for 4 hours. Monocytes were perfused at a constant
flow rate of 1.5 dyn/cm2 after pretreatment with or without
MoAbs to 4 and 2, 8-73 GRO- , or 9-76 MCP-1 peptide analogues
(1 µg/mL each) for 30 minutes on ice before being perfused. (A) The
firm attachment of monocytes to HUVEC was determined by counting the
number of cells attached per field after a 5-minute period. (B) Cells
undergoing spreading or transmigration were counted after 5 minutes and
expressed as the percentage of cells initially attached. (C) The number
of rolling cells within the field was counted in the last 30 seconds of
the 5-minute period and was expressed as the percentage of cells
interacting with endothelium. HUVEC were pretreated with a blocking
(G1) or nonblocking P-selectin (S12) MoAb for 30 minutes at 37°C.
Data are the mean ± SD of at least three independent experiments.
*P < .05 versus uninfected control. **P < .05 versus rAd.I B- -infected HUVEC.
|
|
We next investigated the effect of I B- overexpression on
selectin-mediated rolling interactions of monocytes on endothelium. Previous reports have demonstrated that rolling of monocytes on activated endothelium is largely mediated by interactions of L-selectin and P-selectin.2,3 On TNF- -treated HUVEC, the rolling
fraction of cells was found to be 21.2% ± 6.1% of cells in the
field (Fig 7C). Because initial rolling is rapidly converted into
integrin-mediated firm adhesion, cells were pretreated with MoAbs to
4 and 2 integrins to inhibit arrest and facilitate analysis of
rolling. This resulted in an increase in the rolling fraction of cells,
thus serving as an inverse measure of firm arrest. Overexpression of
I B- in HUVEC followed by TNF- stimulation similarly increased
the rolling fraction of cells (34.5% ± 5.8% of cells in the
field). Preincubation of rAd.I B- -infected HUVEC with a blocking
MoAb to P-selectin caused a clear reduction in the rolling
fraction of cells (Fig 7C) and consequently an inhibition in firm
arrest (47 ± 14 cells/mm2). In contrast, the
nonblocking P-selectin MoAb had no effect on rolling or firm arrest.
Thus, these results show that P-selectin is involved in
monocyte rolling, a prerequisite for firm adhesion.
 |
DISCUSSION |
We have found that overexpression of the cytoplasmic NF- B inhibitor
I B- suppressed the TNF- -stimulated upregulation of ICAM-1,
VCAM-1, and E-selectin and production of GRO- and MCP-1 in
endothelial cells. This was associated with a decrease in the adhesion
and transmigration but not rolling of monocytes on activated endothelium in physiological flow and expands on previous findings that
adenoviral gene transfer of I B- effectively inhibits
NF- B-dependent processes20 in a functionally relevant
system. Whereas TNF- stimulation of uninfected HUVEC markedly
reduced the levels of I B- protein, most likely due to its
proteolytic degradation,15-18 stimulation of
rAd.I B- -infected cells with TNF- decreased the levels of
I B- that, however, remained higher than in control cells.
I B- expressed by adenoviral gene transfer may be phosphorylated and thus more susceptible to TNF- -induced degradation; however, as
observed with LPS stimulation, the overwhelming proportion of I B-
could not be processed.20 In comparison with the adenoviral techniques used here, retrovirus-mediated transfer of wild-type or a
truncated form of I B- did not result in a marked overexpression in HUVEC.22 This may be due in part to the presence of the
nuclear localizing sequence in the adenoviral vector, but also reflects the high efficacy of infection by the adenovirus.
To our best knowledge, this is the first study demonstrating the
effects of I B- overexpression on leukocyte adhesion and transmigration in shear flow. As such, it clearly extends previous reports describing pharmacological and genetic approaches that inhibit
NF- B mobilization or increase the levels of
I B- .19-22,51,52 As previously
found,18,20,21 we show that inhibition of NF- B mobilization by overexpression of I B- inhibited the induction of
endothelial adhesion molecule mRNA and protein expression by TNF- ,
in particular that of the integrin ligands ICAM-1 and VCAM-1. This was
not due to apoptosis, because cell viability was unaltered after
stimulation with TNF- for 4 hours, whereas the onset of apoptosis
occurs only after 6 hours.38 The effects were associated with a significant reduction in the ability of monocytic cells to
firmly arrest and an increase in the rolling fraction of cells on
rAd.I B- -infected HUVEC under physiological flow conditions. It
has been shown that a combination of blocking 4 and 2 integrins inhibits the adhesion of monocytes2,3 and as an inverse
measure of firm arrest, resulted in a shift to more rolling
interactions on activated HUVEC in shear flow. Thus, the effects of
I B- overexpression are likely due to reduced expression of VCAM-1
and ICAM-1, ligands for the 4 integrin VLA-4 and the 2 integrin
LFA-1, respectively. In addition, subsequent spreading and
transmigration of monocytes was reduced on rAd.I B- -infected
HUVEC. Because transendothelial migration of leukocytes requires the
coordinated regulation of integrin avidity, particularly that of VLA-4
and LFA-1,29,53 impaired expression of their ligands may
also contribute to decreased transmigration across
rAd.I B- -infected HUVEC.
Inhibition of NF- B also suppressed the production of the chemokines
MCP-1 and GRO- in TNF- -stimulated HUVEC, which are critically involved in monocyte adhesion and
chemotaxis.14,44-46 Interestingly, overexpression of
I B- appeared to be slightly more effective in inhibiting firm
monocyte arrest on endothelium. Recent reports have shown that CXC
chemokines IP10 and Mig mediate rapid and shear-resistant arrest of T
effector cells, whereas blocking the eotaxin receptor CCR3 in
combination with an 4 MoAb inhibited eosinophil arrest on activated
endothelium.40,48 GRO- can be immobilized to
mmLDL-treated HUVEC and facilitate monocyte adhesion to
endothelium,10 whereas monocyte transmigration appears to
require a soluble gradient of MCP-1.47 Indeed, we have
recently established a hierarchical involvement of chemokines in
monocyte traffic on activated endothelium in shear flow, ie, monocyte
arrest requires surface-bound GRO- , whereas subsequent transmigration is mediated by MCP-1 presented in a soluble gradient created by removal of secreted protein in shear flow.49 We
have confirmed here the contributions of these chemokines using peptide antagonists. Moreover, the combination of MoAb to integrins with peptide antagonists or with rAd.I B- -infection of HUVEC appeared to slightly accentuate the inhibition of monocyte arrest, as well as
spreading and transmigration. Thus, the reduction in GRO- due to
NF- B inhibition may contribute to decreased monocyte adhesion in
shear flow. In addition, overexpression of I B- in HUVEC may result in a marked reduction in MCP-1 secretion that would impair the
establishment of an MCP-1 gradient and thus monocyte transmigration. Hence, our data for the first time provide evidence that, in addition to effects on endothelial Ig integrin ligands, the reduced expression of specifically required chemokines in rAd.I B- -infected HUVEC contributes to the inhibition of monocyte arrest and transmigration in
shear flow.
Interestingly, overexpression of I B- decreased firm adhesion but
increased the rolling fraction of cells, indicating a shift to more
rolling interactions. Blocking MoAb inhibition assays have shown that
monocyte rolling on activated endothelium is largely mediated by P- and
L-selectin, whereas no role for E-selectin was seen,2,3
suggesting that interactions of P- or L-selectin are sufficient.
Although effects of NF- B mobilization on E-selectin expression are
well described,11,18,39 the regulation of P-selectin expression by the NF- B/I B- autoregulatory loop is not yet
clear. Evidence with respect to the upregulation of P-selectin
expression by TNF- is not entirely conclusive.2,54,55
Moreover, proteasome inhibitors and antioxidants have been shown to
decrease the expression of endothelial P-selectin independently of
NF- B inhibition.55 We found that rolling interactions of
monocytes were not affected by NF- B inhibition in endothelial cells
and that P-selectin, which was not inhibited by overexpression of
I B- , plays a partial role in mediating monocyte rolling. These
results are consistent with previous reports showing that rolling is
not affected by E-selectin.2,3
Inhibition of NF- B activation in endothelial cells has been
suggested to be useful in dampening inflammatory
responses.19-22 Notably, the inhibition of endothelial
VCAM-1 and MCP-1 expression that can be specifically detected in
atherosclerotic lesions4,5,9 appeared to be more prominent
than that of ICAM-1, indicating a selective rather than general
immunomodulatory effect. This was consistent with findings with PDTC or
aspirin39,51 and may be due to differential contributions
of a variant NF- B site or dimer compositions to ICAM-1
transcription.13 Adenoviral gene transfer of I B- may
offer advantages in its localized applicability,23-25 but
deleterious effects of inhibiting NF- B translocation in large cell
populations cannot be excluded. Previous reports have confirmed the
efficacy of adenoviral gene transfer in normal, atherosclerotic, or
balloon-injured blood vessels.23-25 Although infection with control adenovirus alone did not increase ICAM-1 expression or cell
arrest in our assays, adenovirus-mediated gene transfer itself may
result in an inflammatory response and antigenicity,56
which warrants further studies addressing the feasibility of adenoviral gene transfer in vivo. In conclusion, overexpression of I B- in
endothelium may reduce extravasation of monocytes and other leukocyte
subsets and be of potential use in limiting the inflammatory response
in areas of atherosclerosis or after balloon injury.
 |
ACKNOWLEDGMENT |
The authors are grateful to Drs I. Clark-Lewis, R. Lobb, R. McEver, and
R. Rothlein for providing valuable reagents and to Prof P.C. Weber for
his continuing support.
 |
FOOTNOTES |
Submitted October 14, 1998; accepted January 26, 1999.
Supported by Deutsche Forschungsgemeinschaft (We-1913/2) and August
Lenz-Stiftung.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Kim S.C. Weber, MD, Institut für
Prophylaxe und Epidemiologie der Kreislaufkrankheiten,
Ludwig-Maximilians Universität, Pettenkoferstrasse 9, D-80336
Munich, Germany; e-mail: kim.weber{at}klp.med.uni-muenchen.de.
 |
REFERENCES |
1.
Ross R:
The pathogenesis of atherosclerosis: A perspective for the 1990s.
Nature
362:801, 1993[Medline]
[Order article via Infotrieve]
2.
Luscinskas FW, Ding H, Tan P, Cumming D, Tedder TF, Gerritsen ME:
L- and P-selectins, but not CD49d (VLA-4) integrins, mediate monocyte initial attachment to TNF- -activated vascular endothelium under flow in vitro.
J Immunol
156:326, 1996
3.
Kukreti S, Konstantopoulus K, Smith CW, McIntire LV:
Molecular mechanisms of monocyte adhesion to IL-1 -stimulated endothelial cells under physiologic flow conditions.
Blood
89:4104, 1997[Abstract/Free Full Text]
4.
Poston RN, Haskard DO, Coucher JR, Gall NP, Johnson-Tidey RR:
Expression of intercellular adhesion molecule-1 in atherosclerotic plaques.
Am J Pathol
140:665, 1992[Abstract]
5.
O'Brien KD, McDonald TO, Chait A, Allen MD, Alpers CE:
Neovascular expression of E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 in human atherosclerosis and their relation to intimal leukocyte content.
Circulation
93:672, 1996[Abstract/Free Full Text]
|