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B-Independent Mechanism1
Department of Cardiothoracic Surgery, National Heart and Lung Institute, Imperial College School of Medicine, Heart Science Centre, Harefield Hospital, Harefield, Middlesex, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B activity,
in contrast to TNF stimulation. Transcription of VCAM-1 mRNA was
observed by reverse-transcriptase PCR after ICAM-1 cross-linking, with
no associated transcription of E-selectin. This was reflected by the
presence of VCAM-1 protein after immunoprecipitation, without
E-selectin expression, in ICAM-1 cross-linked cells. In contrast, mRNA
and protein for both VCAM-1 and E-selectin were observed in TNF-treated
HUVEC, as expected. Addition of the MEK (MAP/Erk kinase)
inhibitor PD98059 reduced expression of VCAM-1 after ICAM-1
cross-linking, suggesting that the Erk pathway is involved in
ICAM-1-mediated VCAM-1 expression. In conclusion, ICAM-1-induced
expression of VCAM-1 represents a pathway for adhesion molecule
up-regulation that is distinct from the TNF-induced pathway. It may be
similar to the IL-4 pathway or it may represent a novel
pathway. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and IL-1 15 . ICAM-1 plays a role in inflammatory processes and in the T cell-mediated host defense system. Within the endothelium, ICAM-1 has an important role in migration of leukocytes to sites of inflammation, enabling the firm adhesion and diapedesis of leukocytes via its interaction with LFA-1 and Mac-1 16, 17 . Its role as an adhesive molecule was initially thought to be the only function of ICAM-1. Recently, however, evidence has emerged to suggest that it can also transduce signals. Using cross-linked Abs to mimic its interaction with its ligands, ligation of ICAM-1 on the cell surface has been shown to induce tyrosine phosphorylation of the cytoskeletal protein cortactin, in rat brain endothelial cells 18 , and tyrosine phosphorylation of the cell cycle protein, cdc2 kinase, in T cells 19 . ICAM-1 cross-linking has also been shown to induce an oxidative burst in PBMC 20 and activation of the AP-13 transcription factor complex, leading to increased IL-1ß production in synovial cells isolated from the rheumatoid joint 21 .
It is well established that signaling through TNF and IL-1 receptors on
endothelial cells leads to induction of VCAM-1 and E-selectin at the
cell surface 22, 23, 24 , through well-defined signal transduction
pathways. NF-B binding sites in the promoters of both molecules have
been shown to be essential for TNF-induced expression of these
molecules 25, 26, 27, 28 . VCAM-1 is a 110-kDa cell adhesion molecule
29, 30, 31 , which is a receptor for 
4ß1
integrin (VLA-4) expressed on the surface of activated mononuclear
cells (monocytes, T cells, and eosinophils) 32, 33 . Its expression on
the endothelium has been shown to increase adhesiveness 34, 35 and
migration of these VLA-4-positive subsets 36, 37 . In this study, we
show that ligation of ICAM-1 on the surface of resting human
endothelial cells leads to the expression of VCAM-1 with no increase in
synthesis of E-selectin mRNA or protein, utilizing signaling cascades
distinct from those initiated after stimulation with TNF.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HUVEC were isolated, as described previously 38 , and maintained in M199 with 2 mM L-glutamine (Life Technologies, Paisley, U.K.), 150 U/ml penicillin/streptomycin (Life Technologies), 20% FCS (Sigma, Poole, U.K.), and 10 ng/ml heparinized endothelial cell growth factor (Boehringer Mannheim, Lewes, U.K.) on gelatin (Sigma)-coated tissue culture flasks (Helena Biosciences, Sunderland, U.K.). Confluent monolayers of single isolates at between passages 4 and 6 were used for all experiments. All experiments were performed with cells from at least three separate isolates.
Antibodies
Protein G-purified fractions of anti-ICAM-1 clone 6.5B5 39
were used at 15 µg/ml. Protein G-purified fractions of
anti-VCAM-1 clone 1.4C3 39 were used at 250 ng/ml for blotting
and 2.5 µg/ml for immunoprecipitation. Anti-VCAM-1 mAb BBIG-V1 and
anti-E-selectin mAb BBIG-E-1 were purchased from R&D Systems
(Abingdon, U.K.) and used at 10 µg/ml for blotting and
immunoprecipitation. Anti-active Erk-1 (Promega, Southampton, U.K.) was
used at 1:10,000 for blotting. Anti-Erk-1 (Transduction Laboratories;
purchased from Affiniti Biosystems, Exeter, U.K.) was used at 4 µg/ml
for immunoprecipitation and 2 µg/ml for blotting. Anti-IB
(Santa Cruz; purchased from Insight Biotechnology, London, U.K.) was
used at 100 ng/ml for blotting. Rabbit anti-mouse Ig Ab Z0259 (RAM)
(Dako, Cambridge, U.K.) was used for cross-linking at 1:100 from
manufacturer’s stock. Goat anti-murine and goat anti-rabbit
horseradish peroxidase-conjugated secondary Abs were purchased from
Jackson (Stratech, Luton, U.K.) and were used at 1:5000, according to
the manufacturer’s instructions.
Western blot analysis and immunodetection
HUVEC were plated onto 9-cm dishes. Once confluent, the cells were washed in serum-free M199 and then incubated in M199 with 5% FCS for 18 h to minimize activation by serum. Immediately before use, the medium was removed and the cells were washed once in serum-free M199 before being incubated in 2 ml M199 with 5% FCS. Anti-ICAM-1 Ab was added as appropriate, and the cells were incubated for 30 min at 37°C. Cells were washed once with serum-free M199 and incubated at 37°C in 2 ml M199 with 5% FCS containing RAM at a dilution of 1/100. Alternatively, HUVEC were incubated with 100 U/ml human rTNF (Genzyme, West Malling, U.K.) for 30 min at 37°C. Monolayers were washed twice in PBS, and cells were lysed in 250 µl RIPA buffer (20 mM MOPS (pH 7), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS) for 10 min on ice. Lysates were removed from the dishes using a rubber policeman and pushed through a small-bore needle several times. Debris was pelleted and supernatants were decanted to fresh tubes. Protein concentration was assayed using a BCA protein assay kit (Pierce and Warriner, Chester, U.K.).
A total of 25 µg protein was electrophoresed on 12% polyacrylamide gels and blotted onto nitrocellulose membrane (Amersham International, Amersham, U.K.). After blocking in 5% nonfat milk, PBS, and 0.01% Tween-20 (marvel PBS-T), membranes were incubated in primary Abs for 1 h with agitation, followed by three washes in PBS-T, each for 5 min. The membranes were incubated with appropriate secondary Abs conjugated to horseradish peroxidase, and after three washes in PBS-T, incubated in enhanced chemoluminescence (ECL) reagents (Amersham) and exposed to autoradiography film (Amersham).
Immunoprecipitation
Cells were stimulated and lysed, as described above. A total of 100 µg of each cell lysate was incubated with primary Ab and 20 µl protein G-Sepharose slurry (Sigma) for 18 h at 4°C with mixing. Beads were pelleted and washed four times in RIPA buffer, before being resuspended in 100 µl SDS gel-loading buffer without reducing agents. The beads were boiled for 5 min and pelleted again, and the supernatants were electrophoresed on 12% polyacrylamide gels, as described above.
Electrophoretic mobility shift assays
HUVEC were plated onto 9-cm dishes and allowed to become confluent. They were allowed to quiesce and stimulated as described above. After treatment, cell lysates were prepared as described previously 40 . Briefly, cells were washed twice in PBS and scraped from the dishes. Cells were pelleted and resuspended in 500 µl lysis buffer A (10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.1% Nonidet P-40, 0.5 mM PMSF, 1 mM EDTA, TPCK (L-1-tosylamido-2-phenylethyl chloromethyl ketone), pepstatin A, and antipain). After 10-min incubation on ice, nuclei were pelleted and resuspended in 20 µl lysis buffer B (20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 0.42 M NaCl, 25% glycerol, and protease inhibitors) and incubated on ice for 10 min. Debris was pelleted, the supernatant was diluted in 80 µl buffer C (20 mM HEPES (pH 7.9), 50 mM KCl, 0.5 mM DTT, 0.2 mM EDTA, 0.5 mM PMSF), and protein concentration was assayed.
A total of 1 µg each nuclear extract was incubated with 0.175 pmol
[-32P]dCTP-labeled complementary oligonucleotides to
the consensus sequence of either NF-B (5'-AGT TGA GGG GAC TTT CCC
AGG C; 5'-TCA ACT CCC CTG AAA GGG TCC G) or AP-1 (5'-CGC TTG ATG AGT
CAG CCG GAA; 5'-GCG AAC TAC TCA GTC GGC CTT) DNA binding sites
(Promega), in a reaction mix containing 10 mM Tris-HCl (pH 7.5), 20 mM
NaCl, 1 mM EDTA, 5% glycerol, 1.5 pmol irrelevant oligodeoxynucleotide
(5'-ATT GTG TAA CTT TTC ATC AGT TGC) 41 , and 2.5 ng poly(dI · dC)
in a final volume of 20 µl, with or without prior addition of a
10-fold excess (1.75 pmol) of unlabeled consensus complementary
oligodeoxynucleotides. Reactions were incubated for 1 h at room
temperature. A total of 2 µl loading buffer (250 mM Tris-HCl (pH
7.5), 0.2% bromophenol blue, 40% glycerol) was added, and reactions
were electrophoresed on prerun 6% native polyacrylamide gels. Gels
were fixed in 10% methanol, 10% glacial acetic acid for 5 min,
wrapped in Saran wrap, and exposed to phosphoimage screens (Kodak,
Rochester, NY) for 3 h. Screens were developed using a Storm 860
PhosphorImager analyzer (Molecular Dynamics, Sunnyvale, CA), and
analysis was performed using Image Quant analysis software (Molecular
Dynamics).
JNK-1 immune complex kinase assays
JNK-1 immune complex kinase assays were conducted as described
previously 42 . Briefly, cells were treated as described above, lysed
with RIPA lysis buffer, and JNK was immunoprecipitated with 10 µl
each of anti JNK-1 Abs Sak-9 and Sak-10 (a kind gift of Prof. J.
Saklatvala, Kennedy Institute of Rheumatology, London, U.K.).
Protein G-Sepharose beads were pelleted and washed twice with cold RIPA
buffer, followed by two washes with kinase buffer (25 mM HEPES (pH
7.3), 25 mM ß-glycerophosphate, 25 mM MgCl2, 0.1% DTT, 1
mM sodium orthophosphate). The beads were then resuspended in kinase
buffer containing 0.37 MBq [-32P]ATP, 20 µM ATP, and
3 µg GST-c-Jun fusion protein (a kind gift of Prof. J. Saklatvala),
and incubated for 30 min at room temperature with vigorous mixing. SDS
gel-loading buffer was added to the samples, and they were boiled for 5
min before separation by 15% SDS-PAGE. Gels were fixed for 5 min, and
exposed to phosphoimage screens for 24 h before developing using a
Storm 860 PhosphorImager analyzer.
Erk-1 immune complex kinase assays
Erk-1 immune complex kinase assays were conducted as described
previously 43 . Briefly, cells were treated as described above, lysed
with RIPA lysis buffer, and Erk-1 was immunoprecipitated with 1 µg
anti Erk-1 Ab. Protein G-Sepharose beads were pelleted and washed twice
with cold RIPA buffer, followed by two washes with 1x kinase buffer.
The beads were then resuspended in 50 µl kinase buffer containing
0.37 MBq [-32P]ATP, 20 µM ATP, and 3 µg of a
peptide of myelin basic protein (Calbiochem, Nottingham, U.K.), and
incubated for 30 min at room temperature with vigorous mixing. After
this time, the reaction was stopped with 15 µl glacial acetic acid,
and endogenous proteins were precipitated with an equal volume of 10%
TCA. A total of 10 µl each sample was spotted onto p81
phosphocellulose discs (Life Technologies). The discs were washed in a
large volume of 0.75 M phosphoric acid and counted on a Canberra
Packard beta counter without scintillant.
RT-PCR
HUVEC were plated onto six-well dishes and allowed to reach confluence. Cells were allowed to quiesce in M199 with 5% FCS for 18 h. Cells were washed in serum-free M199 and replaced with 1 ml M199 with 5% FCS. Anti-ICAM-1 Ab was added together with RAM, and cells were incubated at 37°C for 6 h. The medium was removed from each well, and total RNA was extracted from the cells using RNeasy extraction columns (Quiagen, Crawley, U.K.), according to the manufacturer’s instructions.
cDNA synthesis was conducted using 10 U AMV reverse transcriptase
(Promega) and 200 ng oligo(dT) primer (Promega), together with the
manufacturer’s buffer, 20 nmol dNTP, and 12.5 nmol MgSO4
for 1 h at 48°C. cDNA was stored at -20°C. The PCR was
conducted using 10 U Tfl DNA polymerase (Promega). A total of 1 µl of
each cDNA was used per PCR reaction. A total of 25 nmol
oligodeoxynucleotide primers to adhesion molecules 44 and ß-actin
primers 45 was used as previously described. For sequences of
primers, see Table I
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation
Although ICAM-1 cross-linking on the surface of cells of different
lineages has been shown to induce cellular changes, including new
protein synthesis 21, 46 , the exact pathways leading to these changes
have not been fully investigated. Since many of the genes shown to be
induced by cytokine stimulation of endothelial cells contain
NF-B-binding elements within their promoter regions (tissue factor,
ICAM-1, VCAM-1, E-selectin 25, 26, 27, 28, 47, 48), we first investigated
whether ICAM-1 cross-linking on the surface of HUVEC leads to the
depletion of IB in the cytoplasm of the cell, allowing the
translocation of NF-B to the nucleus and subsequent activation.
HUVEC were incubated for 30 min with anti-ICAM-1 alone, with RAM
alone for 1 h, or with anti-ICAM-1, followed by RAM to
cross-link. None of these treatments, including up to 1-h
cross-linking, caused any change in levels of IB in cytoplasmic
extracts (Fig. 1A). In
contrast, treatment of HUVEC with TNF for 30 min caused a complete
depletion of IB. Similar results were obtained using Abs to
detect the presence of IBß (data not shown).
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B DNA binding site was investigated. Again, no changes in
activation were observed between resting and ICAM-1 cross-linked cells,
while TNF treatment caused a large increase in the binding of nuclear
extracts to the NF-B consensus sequence (Fig. 1
B), which
was abrogated by addition of excess unlabeled NF-B consensus
oligodeoxynucleotide (Fig. 1C). ICAM-1 activation leads to AP-1 activation
We next determined whether nuclear extracts from ICAM-1
cross-linked HUVEC were able to bind to 32P-labeled
complementary oligodeoxynucleotides corresponding to the consensus
sequence of the AP-1 DNA binding site, as has previously been shown for
fibroblast-like synovial cells isolated from the rheumatoid joint 21 .
As shown in Fig. 2A, there was
a clear increase in binding of these extracts upon cross-linking.
Quantitation by densitometry showed there to be no effect of
anti-ICAM-1 Ab alone and a small effect of RAM alone. There was an
approximately 3.5-fold increase in binding above untreated cell
activity after cross-linking for 1 h (p =
0.003), as compared with an approximately 4.3-fold increase in binding
to the consensus sequence after TNF treatment of cells
(p = 0.002) (Fig. 2B). This binding
was specific since coincubation of extracts with an excess of unlabeled
AP-1 consensus oligodeoxynucleotides prevented binding, although an
excess of the NF-B consensus oligodeoxynucleotides had no effect
(Fig. 2C).
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Since ICAM-1 cross-linking caused an increase in AP-1 DNA-binding
activity, the roles of two MAPK-like pathways, able to activate this
transcription factor complex, were examined. Using an Ab that
recognized only the active, phosphorylated form of Erk-1, we saw an
increase in phosphorylated Erk-1 after 30 min cross-linking, and this
active form remained present for up to 1 h (Fig. 3A). There was no effect of
anti-ICAM-1 alone or RAM alone on the phosphorylation of Erk-1.
Pretreatment of HUVEC with the specific MEK inhibitor PD98059 49, 50
inhibited the appearance of phosphorylated Erk-1. Erk-1 activation of
these extracts was also investigated using a peptide of myelin basic
protein as a substrate for immunoprecipitated Erk-1. Again, after 30
min of cross-linking, there was an approximately 40% increase in
activation of Erk-1 above untreated cells (p =
0.007), and this could be inhibited by addition of 30 µM PD98059
(Fig. 3B).
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).
In contrast, an approximately fourfold induction was observed after TNF
treatment of these cells and an approximately eightfold induction after
IL-1 treatment.
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To investigate the possibility that ICAM-1 ligation on the surface
of endothelial cells leads to induction of other cell adhesion
molecules, we examined RNA extracts of ICAM-1 cross-linked cells for
the induction of full-length VCAM-1 using specific intron-spanning
oligodeoxynucleotide primers (Table I). No mRNA for VCAM-1 was observed
in resting HUVEC, but after incubation with anti-ICAM-1 alone,
there was some induction of mRNA, and this was increased after
cross-linking (Fig. 5). Treatment of
HUVEC with RAM alone had no effect on VCAM-1 mRNA. Pretreatment of
cells with the MEK inhibitor PD98059 prevented the induction of VCAM-1
mRNA after ICAM-1 cross-linking and reduced VCAM-1 mRNA expression
after TNF treatment. A second inhibitor, proteasome inhibitor I (PSI),
which has been shown to inhibit NF-B-dependent activities 51 , had
no significant effect on ICAM-1-induced VCAM-1 mRNA induction, but
abrogated TNF-induced VCAM-1 induction, suggesting that the two
molecules utilize different pathways to up-regulate VCAM-1 message.
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B binding 27, 28 . As
shown in Fig. 5A, there was no E-selectin mRNA in resting
cells, and it could not be induced by cross-linking of Abs to ICAM-1,
although as expected, TNF treatment did cause up-regulation of
E-selectin mRNA expression. Interestingly, pretreatment of HUVEC with
PD98059 before ICAM-1 cross-linking caused an up-regulation of
E-selectin mRNA. No such effect was observed after pretreatment of
cells with DMSO alone, or after treatment with PD98059 alone (Fig. 5B). This suggests that there are interactions between
PD98059 and ICAM-1 itself, after cross-linking with Ab, or activation
of novel pathways induced by the combination of these reagents, which
cause induction of E-selectin mRNA.
The induction of E-selectin mRNA by TNF was partially inhibited by
PD98059, but completely abrogated by the addition of PSI, confirming
the dependence on NF-B for TNF-induced E-selectin expression.
Since an increase in VCAM-1 mRNA had been observed in these cells after
ICAM-1 cross-linking, we examined whether this induction led to an
increase in expression of VCAM-1 protein. One limitation of mimicking
cell-cell interaction via ICAM-1 and LFA-1, using Abs to cross-link
ICAM-1, was that it proved difficult to examine surface expression of
adhesion molecules by flow cytometry without binding of Abs to the RAM
used to cross-link the cells. Thus, molecules of interest were
immunoprecipitated from whole cell extracts and immunodetection was
conducted after resolution by SDS-PAGE using Abs directed to the same
and different epitopes of VCAM-1. No induction of VCAM-1 was seen after
treatment with anti-ICAM-1 alone or RAM alone, but we observed a
clear increase in the protein levels of VCAM-1 after 12-h cross-linking
of anti-ICAM-1 or after treatment of cells with TNF for 6 h
(Fig. 6A). In contrast,
extracts from these cells showed no increase in E-selectin protein
after immunoprecipitation and Western blotting, while there was, as
expected, an increase after TNF treatment (Fig. 6B).
Pretreatment of cells with PD98059 caused a decrease in VCAM-1 protein
expression after anti-ICAM-1 cross-linking (Fig. 6C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-treated HUVEC with Abs to E-selectin and ICAM-1
together 46 .
In this study, we have shown that ICAM-1 cross-linking on the surface
of resting HUVEC leads to the activation of the Erk-1 kinase cascade
and subsequent AP-1 transcription factor activity. We were, however,
unable to find any increase in NF-B transcription factor activity
after ICAM-1 cross-linking, either by examining the depletion of the
NF-B inhibitor IB in the cytoplasm of cross-linked cells, or
by analyzing the ability of nuclear extracts from similarly
cross-linked cells to bind to a consensus sequence of the NF-B DNA
binding site. In contrast, cells from the same isolates were able to
up-regulate NF-B activity with accompanying depletion of IB
from the cytoplasm, when treated with TNF.
Surprisingly, we were unable to detect any increase in JNK-1 activity
after cross-linking, despite the increase in AP-1 DNA-binding activity
seen. It is possible that other JNK isoforms or the third MAPK-like
pathway, the p38/Hog1 pathway, may be activated during ICAM-1
cross-linking, leading to the observed AP-1 activation. Again, HUVEC
from the same isolates were able to up-regulate JNK-1 activity after
TNF and IL-1 treatment, suggesting that the cells had not lost the
ability to activate this pathway during culture.
mRNA and protein for VCAM-1 were induced after ICAM-1 cross-linking on
the surface of HUVEC, and this induction was inhibited by addition of
the MEK inhibitor PD98059 to the culture medium, before incubation with
Abs to ICAM-1. This finding suggests that the Erk pathway is
involved in ICAM-1-induced VCAM-1 expression. In contrast, no
induction of mRNA or protein for E-selectin was observed after ICAM-1
cross-linking. This was expected from previous findings that binding of
NF-B to the promoter of E-selectin is required for efficient
transcription 27, 28 .
This report gives credence to a recent finding that ligation of ICAM-1 on the surface of TNF-activated endothelial cells and fibroblasts caused an increase in expression of VCAM-1 and ICAM-1 above that seen for cells activated by cytokine alone 52 . That study, however, did not investigate signaling pathways, which anyway will differ from those reported in this study since the cells used here were rested rather than preactivated by cytokine.
It has been well documented that activation of HUVEC with TNF or IL-1 results in the rapid induction of VCAM-1 and E-selectin 22, 23, 24 . All studies using HUVEC as an endothelial model report coordinated expression of these adhesion molecules, so it was unexpected to find that only VCAM-1 was induced after ICAM-1 cross-linking without any accompanying E-selectin expression. The lack of E-selectin expression was not merely a matter of timing, since E-selectin has previously been shown to be up-regulated before VCAM-1 after cytokine stimulation 23 . The cells were clearly still able to respond to proinflammatory stimuli with the production of both E-selectin and VCAM-1, since TNF treatment for 6 h resulted in increases in mRNA and protein for both molecules.
There are, however, precedents for separate induction of VCAM-1 and
other markers of activation in endothelium. IL-4 has been shown to
induce VCAM-1 expression without accompanying E-selectin expression
53 . In other studies, treatment of HUVEC with IL-4 for 48 h led
to induction of VCAM-1 without any tissue factor or
granulocyte-macrophage CSF expression observed 54 . In a macaque
model, IL-4 markedly increased lymphocyte adhesiveness to lymph node
endothelial cells, and this was blocked with the addition of an Ab to
macaque VCAM-1 34 . Dissection of the signaling pathways involved in
IL-4-mediated VCAM-1 expression has shown that there is no induction of
NF-B after IL-4 treatment of endothelial cells 54 . This suggests
that the signaling pathways downstream from ICAM-1 ligation at the cell
surface, leading to VCAM-1 expression, may follow a similar pattern to
those induced by IL-4 leading to VCAM-1 up-regulation in endothelial
cells. Alternatively, this could represent a novel pathway for VCAM-1
induction. Further studies are underway to distinguish between these
possibilities.
Functional analysis of the VCAM-1 promoter has shown that the two
NF-B sites located at -57 and -72 bp are essential for TNF-induced
expression of VCAM-1 25, 26 . In contrast, the AP-1 site located at
-490 bp was shown not to be necessary for VCAM-1 up-regulation after
TNF stimulation. It was, therefore, interesting to find that ICAM-1
cross-linking led to an up-regulation of VCAM-1 protein without NF-B
activation, but together with an increase in AP-1 activity. It is
possible that the AP-1 site in the VCAM-1 promoter is required for the
induction of mRNA after stimulation of cells by agents that do not have
any effect on NF-B activity, such as IL-4 and ICAM-1 cross-linking.
Physiologically, the most likely route for ICAM-1 cross-linking, in
vivo, would occur via interaction with LFA-1 or Mac-1 on the surface of
leukocytes. While all leukocytes have receptors for ICAM-1, only
mononuclear cells (lymphocytes, monocytes, eosinophils) have receptors
for VCAM-1 (VLA-4; 4ß1) 32, 33 . From the
perspective of cell numbers and cell adhesiveness, neutrophils, which
express no VLA-4, are likely to be the first leukocytes to interact
with the vessel walls early in an inflammatory response, via
interaction of activated LFA-1 on their surface with endothelial ICAM-1
16, 55 . The results presented in this study, using Abs to mimic the
interaction of ICAM-1 with its ligands, suggest that leukocyte
interactions with endothelial cells may themselves accelerate adhesion
molecule expression and may alter in a qualitative way the nature of an
inflammatory response. We have shown that ligation of ICAM-1 leads to
VCAM-1 induction, which could allow increased infiltration of the
VLA-4-positive population into tissue after early contact between
neutrophils and the endothelium at the site of an inflammatory lesion.
Contact between monocytes and endothelium has been shown to lead to an
increase in production of TNF by the monocyte population, leading to
induction of VCAM-1 and subsequent migration of T cell populations that
migrated poorly through unactivated endothelium 56 . If cell contact
also induces selective adhesion molecule expression on the endothelium
in a cytokine-independent manner, then this will provide a mechanism
for increasing extravasation to sites of inflammatory lesions, avoiding
the global proinflammatory effects of TNF early in the inflammatory
response.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. M. Rose, Heart Science Centre, Harefield Hospital, Harefield, Middx UB9 6JH, U.K. E-mail address:
3 Abbreviations used in this paper: AP-1, activator protein-1; GST, glutathione-S-transferase; IB, inhibitor of B; JNK, Janus kinase; MAPK, mitogen-activated protein kinase; Erk-1, extracellular signal-regulated kinase-1; MEK, MAP/Erk kinase; PSI, proteasome inhibitor I; RAM, rabbit anti-mouse; VLA, very late antigen.
Received for publication August 10, 1998. Accepted for publication November 30, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B-like factor is essential but not sufficient for cytokine induction of endothelial leukocyte adhesion molecule 1 (ELAM-1) gene transcription. Nucleic Acids Res. 19:2645.4 ß7/4 ßP in lymphocyte adherence to fibronectin and VCAM-1 and in homotypic cell clustering. J. Cell Biol. 117:179. to selectively enhance endothelial cell adhesiveness for T cells: the contribution of vascular cell adhesion molecule-1-dependent and -independent binding mechanisms. J. Immunol. 146:592.[Abstract]
and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor B. Proc. Natl. Acad. Sci. USA 86:2336. action. J. Immunol. 158:1095.[Abstract]
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B/Rel family members regulating the ICAM-1 promoter in monocytic THP-1 cells. Immunobiology 198:50.[Medline]
B. Proc. Natl. Acad. Sci. USA 93:3308. in cultured endothelial cells. J. Clin. Invest. 95:264.
B-independent mechanism. FEBS Lett. 372:194.[Medline]
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 Y.-C. Lin, C.-T. Shun, M.-S. Wu, and C.-C. Chen A Novel Anticancer Effect of Thalidomide: Inhibition of Intercellular Adhesion Molecule-1-Mediated Cell Invasion and Metastasis through Suppression of Nuclear Factor-{kappa}B Clin. Cancer Res., December 1, 2006; 12(23): 7165 - 7173. [Abstract] [Full Text] [PDF]
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 E. Cernuda-Morollon and A. J. Ridley Rho GTPases and Leukocyte Adhesion Receptor Expression and Function in Endothelial Cells Circ. Res., March 31, 2006; 98(6): 757 - 767. [Abstract] [Full Text] [PDF]
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 A. Beklen, M. Laine, I. Venta, T. Hyrkas, and Y.T. Konttinen Role of TNF-{alpha} and Its Receptors in Pericoronitis Journal of Dental Research, December 1, 2005; 84(12): 1178 - 1182. [Abstract] [Full Text] [PDF]
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 S. van Wetering, N. van den Berk, J. D. van Buul, F. P. J. Mul, I. Lommerse, R. Mous, J.-P. t. Klooster, J.-J. Zwaginga, and P. L. Hordijk VCAM-1-mediated Rac signaling controls endothelial cell-cell contacts and leukocyte transmigration Am J Physiol Cell Physiol, August 1, 2003; 285(2): C343 - C352. [Abstract] [Full Text] [PDF]
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 G. Vassalli, A. Gallino, M. Weis, W. von Scheidt, L. Kappenberger, L.K. von Segesser, J.-J. Goy, and on behalf of the Working Group Microcirculation of Alloimmunity and nonimmunologic risk factors in cardiac allograft vasculopathy Eur. Heart J., July 1, 2003; 24(13): 1180 - 1188. [Abstract] [Full Text] [PDF]
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 R. Blaber, E. Stylianou, A. Clayton, and R. Steadman Selective Regulation of ICAM-1 and RANTES Gene Expression after ICAM-1 Ligation on Human Renal Fibroblasts J. Am. Soc. Nephrol., January 1, 2003; 14(1): 116 - 127. [Abstract] [Full Text] [PDF]
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 Q. Wang, G. R. Pfeiffer II, T. Stevens, and C. M. Doerschuk Lung Microvascular and Arterial Endothelial Cells Differ in Their Responses to Intercellular Adhesion Molecule-1 Ligation Am. J. Respir. Crit. Care Med., September 15, 2002; 166(6): 872 - 877. [Abstract] [Full Text]
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 N.-H. Cho, S.-Y. Seong, M.-S. Huh, N.-H. Kim, M.-s. Choi, and I.-s. Kim Induction of the Gene Encoding Macrophage Chemoattractant Protein 1 by Orientia tsutsugamushi in Human Endothelial Cells Involves Activation of Transcription Factor Activator Protein 1 Infect. Immun., September 1, 2002; 70(9): 4841 - 4850. [Abstract] [Full Text] [PDF]
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 C. D. Raeburn, C. M. Calkins, M. A. Zimmerman, Y. Song, L. Ao, A. Banerjee, A. H. Harken, and X. Meng ICAM-1 and VCAM-1 mediate endotoxemic myocardial dysfunction independent of neutrophil accumulation Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2002; 283(2): R477 - R486. [Abstract] [Full Text] [PDF]
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 P. W. Thompson, A. M. Randi, and A. J. Ridley Intercellular Adhesion Molecule (ICAM)-1, But Not ICAM-2, Activates RhoA and Stimulates c-fos and rhoA Transcription in Endothelial Cells J. Immunol., July 15, 2002; 169(2): 1007 - 1013. [Abstract] [Full Text] [PDF]
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A. M. Joussen, V. Poulaki, A. Tsujikawa, W. Qin, T. Qaum, Q. Xu, Y. Moromizato, S.-E. Bursell, S. J. Wiegand, J. Rudge, et al. Suppression of Diabetic Retinopathy with Angiopoietin-1 Am. J. Pathol., May 1, 2002; 160(5): 1683 - 1693. [Abstract] [Full Text] [PDF]
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M. Yasuda, S. Shimizu, K. Ohhinata, S. Naito, S. Tokuyama, Y. Mori, Y. Kiuchi, and T. Yamamoto Differential roles of ICAM-1 and E-selectin in polymorphonuclear leukocyte-induced angiogenesis Am J Physiol Cell Physiol, April 1, 2002; 282(4): C917 - C925. [Abstract] [Full Text] [PDF]
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 E. P. Moiseeva Adhesion receptors of vascular smooth muscle cells and their functions Cardiovasc Res, December 1, 2001; 52(3): 372 - 386. [Abstract] [Full Text] [PDF]
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C. G. Kevil, R. P. Patel, and D. C. Bullard Essential role of ICAM-1 in mediating monocyte adhesion to aortic endothelial cells Am J Physiol Cell Physiol, November 1, 2001; 281(5): C1442 - C1447. [Abstract] [Full Text] [PDF]
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Q. Wang and C. M. Doerschuk The p38 Mitogen-Activated Protein Kinase Mediates Cytoskeletal Remodeling in Pulmonary Microvascular Endothelial Cells Upon Intracellular Adhesion Molecule-1 Ligation J. Immunol., June 1, 2001; 166(11): 6877 - 6884. [Abstract] [Full Text] [PDF]
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C. Lawson, M. E Ainsworth, A. M McCormack, M. Yacoub, and M. L Rose Effects of cross-linking ICAM-1 on the surface of human vascular smooth muscle cells: induction of VCAM-1 but no proliferation Cardiovasc Res, June 1, 2001; 50(3): 547 - 555. [Abstract] [Full Text] [PDF]
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U. G. Strauch, R. C. Mueller, X. Y. Li, M. Cernadas, J. M. G. Higgins, D. G. Binion, and C. M. Parker Integrin {{alpha}}E(CD103){{beta}}7 Mediates Adhesion to Intestinal Microvascular Endothelial Cell Lines Via an E-Cadherin-Independent Interaction J. Immunol., March 1, 2001; 166(5): 3506 - 3514. [Abstract] [Full Text] [PDF]
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S. J. Lee, K. Drabik, N. J. Van Wagoner, S. Lee, C. Choi, Y. Dong, and E. N. Benveniste ICAM-1-Induced Expression of Proinflammatory Cytokines in Astrocytes: Involvement of Extracellular Signal-Regulated Kinase and p38 Mitogen-Activated Protein Kinase Pathways J. Immunol., October 15, 2000; 165(8): 4658 - 4666. [Abstract] [Full Text] [PDF]
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Y. Hu, J.-M. Kiely, B. E. Szente, A. Rosenzweig, and M. A. Gimbrone Jr. E-Selectin-Dependent Signaling Via the Mitogen-Activated Protein Kinase Pathway in Vascular Endothelial Cells J. Immunol., August 15, 2000; 165(4): 2142 - 2148. [Abstract] [Full Text] [PDF]
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Q. Wang and C. M. Doerschuk Neutrophil-Induced Changes in the Biomechanical Properties of Endothelial Cells: Roles of ICAM-1 and Reactive Oxygen Species J. Immunol., June 15, 2000; 164(12): 6487 - 6494. [Abstract] [Full Text] [PDF]
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K. A. Kelly, J. C. Walker, S. H. Jameel, H. L. Gray, and R. G. Rank Differential Regulation of CD4 Lymphocyte Recruitment between the Upper and Lower Regions of the Genital Tract during Chlamydia trachomatis Infection Infect. Immun., March 1, 2000; 68(3): 1519 - 1528. [Abstract] [Full Text] [PDF]
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