Monocyte Adherence Induced by Lipopolysaccharide Involves
CD14, LFA-1, and Cytohesin-1
REGULATION BY Rho AND PHOSPHATIDYLINOSITOL 3-KINASE*
Zakaria
Hmama
§,
Keith L.
Knutson
,
Patricia
Herrera-Velit
,
Devki
Nandan
, and
Neil E.
Reiner
¶
From
Department of Medicine (Division of Infectious
Diseases) and the ¶ Department of Microbiology and Immunology, The
University of British Columbia, Faculties of Medicine and Science, The
Research Institute of the Vancouver Hospital and Health Sciences
Center, Vancouver, British Columbia V5Z 3J5, Canada and
§ The Laboratoire d'Immunologie, Faculté des Sciences
Dhar Mahraz, Université Mohamed Ben Abdallah, BP 1796, Atlas
Fés, Morocco
 |
ABSTRACT |
Mechanisms regulating lipopolysaccharide
(LPS)-induced adherence to intercellular adhesion molecule (ICAM)-1
were examined using THP-1 cells transfected with CD14-cDNA
(THP-1wt). THP-1wt adherence to ICAM-1 was LPS dose-related,
time-dependent, and inhibited by antibodies to either CD14
or leukocyte function associated antigen (LFA)-1, but was independent
of any change in the number of surface expressed LFA-1 molecules. A
potential role for phosphatidylinositol (PI) 3-kinase (PI 3-kinase) in
LPS-induced adherence was examined using the PI 3-kinase inhibitors
LY294002 and Wortmannin. Both inhibitors selectively attenuated
LPS-induced, but not phorbol 12-myristate 13-acetate-induced adherence.
Inhibition by these agents was unrelated to any changes in either LPS
binding to or LFA-1 expression by THP-1wt cells. LPS-induced adherence
was also abrogated in U937 cells transfected with a dominant negative
mutant of of PI 3-kinase. Toxin B from Clostridium
difficile, an inhibitor of the Rho family of GTP-binding
proteins, abrogated both PI-3 kinase activation and adherence induced
by LPS. Cytohesin-1, a phosphatidylinositol
3,4,5-triphosphate-regulated adaptor molecule for LFA-1 activation, was
found to be expressed in THP-1wt cells. In addition, treatment of
THP-1wt with cytohesin-1 antisense attenuated LPS-induced adherence.
These findings suggest a model in which LPS induces adherence through a
process of "inside-out" signaling involving CD14, Rho, and PI
3-kinase. This converts low avidity LFA-1 into an active form capable
of increased binding to ICAM-1. This change in LFA-1 appears to be
cytohesin-1-dependent.
 |
INTRODUCTION |
Adherence of monocytes to endothelial cells is an essential
requirement for the localization of these cells to sites of tissue inflammation (1-3). Several reports have shown that this process is
dependent upon the monocyte surface molecule lymphocyte
function-associated antigen-1
(LFA-1)1 (CD11a/CD18;
L
2) (Refs. 3-5 and reviewed in Refs. 6
and 7). Intercellular adhesion molecule-1 (ICAM-1) (CD54) has been identified as a high affinity counter-receptor for LFA-1 (8). Interactions of ICAM-1 with LFA-1 mediate several important functions in the immune system in addition to adherence (6). The basal affinity
of LFA-1 for ICAM-1 or its other ligands is low and LFA-1 must be
activated to mediate stable adhesion (4, 5). Indeed, in its activated
form, the affinity of LFA-1 for ICAM-1 increases 200-fold in comparison
with its affinity in the resting state (9). This is consistent with a
process of "inside-out" signaling that converts LFA-1 into an
activated form capable of mediating increased adhesion. It is important
to note that conditions which give rise to increased adherence do not
necessarily lead to increased cell surface expression of LFA-1 (5,
10).
The signaling events that link cell stimulation to the activation of
LFA-1 are incompletely understood. Recently, a regulatory protein that
interacts with the cytoplasmic tail of CD18 has been cloned (11). This
protein, cytohesin-1, contains a pleckstrin homology domain that binds
the phosphatidylinositol 3-kinase (PI 3-kinase) metabolite,
phosphatidylinositol 3,4,5-triphosphate (PtdIns-3,4,5-P3),
leading to changes in properties of the protein (12). These findings
suggest a potential role for PI 3-kinase in regulating the activity of
LFA-1.
Bacterial lipopolysaccharide (LPS) is known to enhance the accumulation
of leukocytes at inflammatory foci (13) and the adherence of leukocytes
to endothelial cells in vitro (14, 15). Although it has also
been shown that LPS-induced adherence is mediated at least in part by
LFA-1 (16), the pathway linking LPS to LFA-1 has not been
characterized. A dominant LPS signaling pathway involves the membrane
receptor CD14 (17, 18). Binding of LPS to CD14 results in the
activation of multiple Src family protein tyrosine kinases, and this
appears to involve the physical association of p53/p56lyn with
the receptor (19). It has also been shown that LPS induces the
CD14-dependent association of an activated form of PI
3-kinase with p53/p56lyn (20). Furthermore, activation of
monocyte PI 3-kinase by LPS results in the generation of
PtdIns-3,4,5-P3 (21), which as discussed above is known to
regulate various effector functions including cell adhesion (22, 23).
Taken together, these findings suggest the possibility that LPS-induced
adhesion may be mediated through a pathway involving PI 3-kinase
leading to changes in LFA-1 activity. The results of the present study
show that LPS binding to CD14 induces monocyte adherence dependent upon
LFA-1, ICAM-1, and cytohesin-1, via a PI
3-kinase-dependent pathway regulated by the small
GTP-binding protein Rho.
 |
EXPERIMENTAL PROCEDURES |
Reagents and Chemicals--
RPMI 1640, Hanks' balanced salt
solution (HBSS), and penicillin/streptomycin were from Stem Cell
Technologies (Vancouver, British Columbia). Human AB+ serum
was provided by The Canadian Red Cross (Vancouver, British Columbia).
LPS from Escherichia coli O127:B8 was from Difco. LPS was
labeled with FITC as described (24, 25). Purified soluble, recombinant
ICAM-1 (sICAM-1) was a generous gift from Dr. J. R. Woska, Jr.
(Boehringer Ingelheim, Ridgefield, CT). Phorbol 12-myristate 13-acetate
(PMA), wortmannin,
L-
-phosphatidyl-L-serine),
L-
-phosphatidylinositol (PtdIns), and
L-
-phosphatidylinositol 4,5-diphosphate were purchased from Sigma. LY294002 was from Calbiochem. Toxin B purified from Clostridium difficile was generously provided by Dr. G. Armstrong (University of Alberta, Edmonton, Alberta). Protein
A-agarose, and electrophoresis reagents were purchased from Bio-Rad.
[
-32P]ATP was from Amersham International (Oakville,
Ontario, Canada).
Monoclonal Antibodies (mAbs)--
The following mAbs were used:
3C10 (IgG2b, anti-CD14 mAb, a gift from Dr. W. C. Van Voorhis,
University of Washington, Seattle, WA), W6/32 (IgG2a, anti-HLA-class I,
American Type Culture Collection, Manassas, VA), TS1/18 (IgG1,
anti-CD18), and TS1/22 (IgG1, anti-CD11a) were from the Hybridoma Bank
of the University of Iowa, Iowa city, IA. Anti-PI 3-kinase mAb was from
Upstate Biotechnology (Lake Placid, NY).
Cell Lines--
The monocytic cell lines THP-1wt (THP-1 cells
stably expressing glycosylphosphatidylinositol-anchored CD14) and
THP-1rsv (THP-1 cells transfected with vector alone) were kindly
provided by Dr. R. Ulevitch (The Scripps Research Institute, La Jolla,
CA). Transfection of the promonocytic cell line U937 with cDNA
encoding the entire coding region of either wild-type bovine PI
3-kinase subunit p85
(Wp85
) or mutant bovine p85
(
p85
)
was as described (21). The mutant has a deletion of 35 amino acids from
residues 479-513 of bovine p85
and the insertion of two other amino
acids (Ser-Arg) in the deleted position. This alteration prevents the
association of mutant p85
with the p110 catalytic subunit. Mutant
p85
competes with native p85 for binding to essential signaling
proteins, thereby acting as a dominant negative mutant (21, 26). These
transfected cell lines are referred to as Wp85-U937 and
p85-U937.
All cell lines were cultured in RPMI 1640 supplemented with 10% FCS
(Hyclone, Logan, UT), 2 mM L-glutamine,
penicillin (100 units/ml), and streptomycin (100 µg/ml).
Cell Adhesion to Immobilized sICAM-1--
Flat-bottomed,
polystyrene, cell culture-treated, microtiter plates (Becton Dickinson,
Franklin Lakes, NJ) were loaded with purified sICAM or BSA (coating
control) diluted in 0.1 M carbonate buffer (pH 9) for
1 h at 37 °C and then transferred to 4 °C overnight. Wells
were then washed with HBSS and blocked with 1% BSA in RPMI 1640 for
1 h at 37 °C. Blocking solution was discarded and 100-µl aliquots of cells (1 × 105) were dispensed into
duplicate wells. Plates were incubated for 30 min at 37 °C to allow
cell sedimentation prior to treatment with stimuli or inhibitors. Cells
were then assayed for adherence at 37 °C. Nonadherent cells were
removed by carefully washing three times with 200 µl of warm
(37 °C) incubation medium. Adherent cells were then fixed with 2%
paraformaldehyde/HBSS. Fixed cells were washed once with HBSS and
stained for 10 min with 0.05% crystal violet in 20% methanol. Crystal
violet was rinsed out of the wells with water, and the plates were
allowed to dry. The dye was then eluted from cells by addition of 100 µl of 100% methanol, and absorbance at 570 nm was immediately
recorded in a microtiter plate reader. Adherent cells were quantitated
by using a standard curve generated with a range of input cell numbers
incubated with PMA (20 ng/ml) for 60 min at 37 °C and fixed without
prior washing.
Cell Surface Phenotype Analysis--
To measure the expression
of cell surface molecules, cells were incubated with specific mouse mAb
(10 µg/ml) for 30 min, then washed twice and labeled with
FITC-conjugated F(ab')2 sheep anti-mouse IgG (Sigma) for 30 min. Cells were then washed twice and fixed in 2% paraformaldehyde in
staining buffer. All staining and washing procedures were performed at
4 °C in HBSS containing 0.1% NaN3 and 1% FCS. Cell
fluorescence was analyzed using a Coulter Elite flow cytometer
(Hialeah, FL). Relative fluorescence intensities of 5000 cells were
recorded as single-parameter histograms (log scale, 1024 channels, 4 log decades), and the mean fluorescence intensity (MFI) was calculated
for each histogram. Results are expressed as specific MFI index, which
corresponds to the ratio: MFI of cells + specific antibody/MFI of cells + irrelevant isotype-matched IgG.
In Vitro PI 3-Kinase Assay--
Aliquots of cell lysates
adjusted for protein content (300-500 µg of protein) were incubated
for 4 h at 4 °C with mAb to PI 3-kinase, and immune complexes
were adsorbed onto protein A-agarose for 1 h. The complexes were
washed twice with lysis buffer and three times with 10 mM
Tris-HCl (pH 7.4). PI 3-kinase activity was measured as described (20).
Briefly, immunoprecipitates were incubated for 10 min at 4 °C with
10 µg of sonicated (3 × 20 s in a ultrasonic cell
disrupter, Branson Sonic Power Co., Danbury, CT) PtdIns in 10 µl of
30 mM Hepes to which was added 40 µl of kinase buffer (30 mM Hepes, 30 mM MgCl2, 200 µM adenosine, 50 µM ATP, and 10 µCi of
[
-32P]ATP). Reactions were carried out for 15 min at
room temperature and stopped by the addition of 100 µl of 1 N HCl and 200 µl of chloroform:methanol (1:1, v/v).
Lipids were separated on oxalate-treated silica TLC plates using a
solvent system of chloroform:methanol:water:28% ammonia
(45:35:7.5:2.5, v/v/v/v). Plates were exposed to x-ray film at
70 °C. Incorporation of radioactivity into lipids was quantitated
by excising the corresponding portions of the TLC plate followed by
liquid scintillation counting.
RNA Isolation and RT-PCR--
RNA isolation, cDNA synthesis,
and PCR conditions were as described previously (27). Sequences of
oligonucleotide primers used in PCR amplifications were as follows:
cytohesin-1 sense, CGC GGG GAA TTC GCC ACC ATG GAG GAG GAC GAC AGC TAC
GTT CCC; cytohesin-1 antisense, CGC GGG GCG GCC GCT TTA GTG TCG CTT CGT
GGA GGA GAC CTT (11);
-actin sense, CAC CCC GTG CTG CTG ACC GAG GCC;
-actin antisense, CCA CAC GGA GTA CTT GCG CTC AGG (27). Appropriate negative controls (no DNA and RNA without RT) were included in the
RT-PCR experiments.
Sense and Antisense
Oligonucleotides--
Phosphorothioate-modified oligonucleotides
(S-oligos) to cytohesin-1 were synthesized by Life Technologies, Inc.
Twenty one-mer sequences spanning the presumed translation initiation
site of human cytohesin-1 cDNA (28) were made in both sense and
antisense orientations as follows: sense, 5'-ATG GAG GAG GAC GAC AGC
TAC-3'; antisense, 5'-GTA GCT GTC GTC CTC CTC CAT-3'. The sequences
were selected by screening for uniqueness using Blast 227 and were also
tested for lack of secondary structure and oligonucleotide pairing by
using Primer Software (version 2.0, Scientific and Educational
Software). THP-1wt cells (106) were incubated for 2 h
at 37 °C and 5% CO2 in 250 µl of RPMI 1640 containing
2.5% LipofectAMINE (Life Technologies, Inc.) and various
concentrations of S-oligos. After incubation, the medium was adjusted
to 1 ml and supplemented with 10% FCS, and cells were cultured for an
additional 18 h. Cells were then washed and tested in adhesion
assay as described above.
 |
RESULTS |
THP-1 Phenotype Analysis--
Surface expression of CD14 and LFA-1
molecules was analyzed by immunofluorescence and FACS analysis. The
representative record shown in Fig. 1
demonstrates that cells transfected with CD14 (THP-1wt) and control
cells transfected with vector alone (THP-1rsv) expressed similar levels
of both LFA-1
- and
-chains (CD11a and CD18, respectively).
However, THP-1wt cells expressed about 80-fold more CD14 than did
THP-1rsv cells.

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Fig. 1.
CD14 and LFA-1 surface expression on THP-1
cell lines. THP-1rsv and THP-1wt cells were incubated for 30 min
at 4 °C with either anti-CD14 (a), anti-CD18 (LFA-1
-chain) (b), or anti-CD11a (LFA-1 -chain)
(c), then washed twice and labeled with FITC-conjugated
F(ab)'2 sheep anti-mouse IgG. Samples were washed twice and
fixed in 2% paraformaldehyde before FACS analysis. Results are
expressed as histograms of fluorescence intensity (log scale) derived
from 5000 events. In each panel, histograms displaced to the
right represent cells stained with specific mAbs, and
histograms on the left represent cells stained with
irrelevant isotype-matched IgG. Numerical values in the top
right of each frame indicate the MFI index, which corresponds to
the ratio: MFI of cells incubated with specific antibody/MFI of cells
stained with irrelevant isotype-matched IgG. The data shown are
representative of results obtained in five separate experiments
yielding similar results.
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Adherence of THP-1 Cells to Immobilized sICAM-1-coated
Plates--
Initial experiments were carried out to standardize the
model system of LPS-induced adherence of THP-1 cells to sICAM-1.
CD14-transfected THP-1wt cells and control THP-1rsv cells were
incubated in microtiter wells coated with sICAM-1 at concentrations
ranging from 0 to 40 µg/ml and treated with LPS in the presence of
0.5% normal AB+ serum. THP-1wt cells adhered to sICAM-1 in
a dose-dependent manner with a maximum of 48 ± 8.7%
cells binding to wells coated with 20 µg/ml of sICAM-1 (Fig.
2A). In contrast, control
THP-1rsv cells adhered at maximum rate of 8.3 ± 2%. In the
absence of sICAM-1, only 4-5% of the THP-1wt cell adhered
nonspecifically to the plate (data not shown). Binding specificity for
ICAM-1 was also demonstrated by control experiments in which only
4.8 ± 3.2% of THP-1wt cells adhered to wells coated with BSA at
20 µg/ml (n = 4, data not shown).

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Fig. 2.
THP-1 cell adherence to immobilized
sICAM-1. Flat-bottomed, polystyrene cell culture-treated 96-well
microtiter plates were loaded with 105 cells/well in 200 µl of RPMI 1640 (final volume), and cells were allowed to adhere at
37 °C in a humidified atmosphere containing 5% CO2.
Unbound cells were washed away, and attached cells were fixed with 2%
paraformaldehyde/HBSS and stained with 0.05% crystal violet. Excess
staining solution was rinsed away, and the absorbance of the dye
retained by the adherent cells (eluted by addition of 100% methanol)
was measured in individual wells at 570 nm. Duplicate determinations
were made for each data point. Adherence was quantitated using a
standard curve generated with a range of known input cell numbers.
A, adherence of THP-1wt or THP-1rsv cells in response to 2 µg/ml LPS (in presence of 0.5% AB+) assayed for 1 h
in wells precoated with a range of concentration of sICAM-1 (0-40
µg/ml). B, THP-1wt cells were stimulated with a range of
concentration of LPS (0-10 µg/ml) in presence of 0.5%
AB+ serum for 1 h in wells coated with 20 µg/ml of
sICAM-1 or BSA. C, adherence of THP-1wt and THP-1rsv cells
stimulated with LPS (1 µg/ml in 0.5% AB+ serum) for
different times (7 min to 2 h) in wells coated with 20 µg/ml
sICAM-1. The values shown in each panel are the averages of two
independent determinations obtained in separate experiments.
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THP-1wt and THP-1rsv cells were also examined for adherence in the
presence of a range (0.1-10 µg/ml) of concentrations of LPS (Fig.
2B). Treatment of THP-1wt cells with LPS resulted in a
dose-dependent increase in adherence that was maximal
(52.1 ± 6.1%, mean ± S.E., n = 2) at 1 µg/ml. Maximal adherence observed with THP-1rsv was only 10.7 ± 1.8% (mean ± S.E., n = 2), and this was not
affected by LPS, indicating that LPS-induced adherence in this system
was largely CD14-dependent. In other experiments, cells
were incubated with LPS (1 µg/ml in 0.5% AB+ serum) for
up to 2 h to determine the time course of THP-1 adherence to
sICAM-1 (Fig. 2C). LPS-stimulated adherence was maximal by 60 min and remained stable for the second hour. The importance of serum
was also addressed. The results showed that exposure of cells to LPS in
the absence of serum resulted in markedly reduced adherence (12.1 ± 0.8%, mean ± S.E., n = 2). Conversely,
treatment with serum alone did not induce cell adherence (data not shown).
Cell Surface Molecules Involved in LPS-induced THP-1 Adherence to
sICAM-1--
As shown above, only cells transfected with CD14
displayed enhanced adherence in response to LPS. The role of CD14 in
cell adherence was investigated further by competitive inhibition with a neutralizing anti-CD14 mAb 3C10 (18, 29). Fig.
3A shows that preincubation of
THP-1wt cells with mAb 3C10 prior exposure to LPS led to approximately
80% inhibition of adherence. Competitive inhibition with mAb was also
used to examine the extent of involvement of LFA-1 in this model. The
results obtained (Fig. 3A) show significant inhibition with
either neutralizing anti-CD11a mAb (TS1/18) or anti-CD18 mAb (TS1/22)
(30, 31). When a range of concentrations of anti-CD14 and anti-LFA-1
mAbs were tested, a concentration of 10 µg/ml was observed to result
in maximal inhibition of LPS-induced adherence (data not shown).
Furthermore, when compared with blockade of either CD11a or CD18 alone,
inhibition (85%) was enhanced when anti-CD18 and anti-CD11a were used
in combination. Specificity of inhibition by anti-CD14 and anti-LFA-1
mAbs was validated using anti-MHC class I mAb, W6/32. Despite its
binding to constitutively expressed cell surface molecules, W6/32 did
not affect LPS-induced adherence.

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Fig. 3.
Inhibition of LPS-induced adherence with
anti-CD14 and anti-LFA-1 mAbs (A) and effect of
LPS on cell surface expression of LFA-1 (B).
A, microtiter wells were loaded with THP-1wt cells (1 × 105 in RPMI 1640), and cells were incubated for 30 min
at room temperature with either W6/32 (anti-HLA class I), 3C10
(anti-CD14), TS1/18 (anti-LFA-1 , -chain), TS1/22 (anti-LFA-1 ,
-chain), or a mixture (1:1) of TS1/18 and TS1/22 (final
concentrations, 10 µg/ml). LPS was then added to a final
concentration of 1 µg/ml in 0.5% AB+ serum, and cells
were assayed for adherence for 30 min at 37 °C and 5%
CO2. Adherent cells were stained with crystal violet, and
the absorbances were measured as described in the legend to Fig. 2.
Results are expressed as percent of maximal adherence, which
corresponds to: absorbance of cells incubated with antibody prior to
exposure to LPS/absorbance of cells treated with LPS in the absence of
antibody × 100. B, THP-1rsv cells were incubated for
30 min at 37 °C in medium alone (a), in medium plus 1 µg/ml LPS (0.5% AB+ serum) (b), in medium
plus 5 µg/ml LPS (0.5% AB+ serum) (c), or in
medium plus 5 × 10 7 M fMet-Leu-Phe
(d). Cells were washed and stained with either anti-CD11a
(LFA-1 -chain) or anti-CD18 (LFA-1 -chain) and then with
FITC-conjugated F(ab)'2 sheep anti-mouse IgG as described
in the legend to Fig. 1. In each panel, histograms displaced to the
right represent cells stained with specific mAbs, and
histograms on the left represent cells stained with
irrelevant isotype-matched IgG. Numerical values in boldface
indicate MFI indices calculated as described in the legend to Fig. 1.
The data shown in A are means ± S.E. of values
obtained in three separate experiments. B shows
representative results from one of two separate experiments yielding
similar results.
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Certain agonists are known to induce increased adherence by either
up-regulating integrin expression, by increasing integrin affinity for
substrate or by a combination of these effects (32). To address whether
the difference observed between THP-1wt and THP-1rsv could be related
to altered integrin expression induced by LPS, cell surface expression
of CD11a and CD18 was analyzed. As shown in Fig. 3B, LPS
treatment did not affect the expression of these proteins by THP-1wt
cells. In contrast, incubation of cells with fMet-Leu-Phe, an agonist
known to up-regulate integrin expression (33), induced significant
increases in expression of both CD11a and CD18. Taken together, these
findings suggest that LPS does not modify the number of surface
expressed LFA-1 molecules, but rather initiates a signaling sequence
through CD14 leading to increased avidity of LFA-1 for ICAM-1.
LPS-induced Adherence Is Phosphatidylinositol
3-Kinase-dependent--
Recent evidence has suggested a
role for PI 3-kinase in signaling pathways activated by LPS (20, 21).
To examine the potential involvement of PI 3-kinase in LPS-induced
adherence, cells were incubated with various concentration of the PI
3-kinase inhibitors wortmannin and LY294002 for 20 min prior to the
addition of LPS. Preincubation with wortmannin inhibited LPS-induced
adherence in a dose-dependent manner (Fig.
4A, maximum inhibition 92.9 ± 6.7%, mean ± S.E., n = 3) with an
IC50 of approximately 1 nM. This value is 10 times lower than the IC50 for wortmannin as determined for
inhibition of fMet-Leu-Phe-stimulated neutrophil homotypic aggregation
(33). LY294002, an inhibitor of PI 3-kinase that acts via a distinct
mechanism, when used at concentrations known to be relatively selective
for inhibition of PI 3-kinase, also attenuated LPS-induced adherence
(Fig. 4B, maximum inhibition 89.5 ± 2.1%, mean ± S.E., IC50 ~0.45 µM, n = 3). In contrast to abrogation of LPS-induced adherence, neither
wortmannin nor LY294002 had significant effects on PMA-induced
adherence, except at high concentrations. These results show that
inhibition of LPS-induced adherence by wortmannin or LY294002 is not
due to nonspecific toxicity. Moreover, they suggest that the pathways
regulating adherence in response to PMA and LPS are distinct.

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Fig. 4.
Wortmannin and LY294002 attenuate LPS-induced
adherence. 1 × 105 THP-1wt cells (in 200 µl of
RPMI 1640, final volume) were dispensed into 96-well microtiter plates
and incubated for 20 min at 37 °C and 5% CO2 with
various concentrations of wortmannin (0-100 nM)
(A) or LY294002 (0-32 µM) (B).
Either LPS (1 µg/ml in 0.5% AB+ serum) or PMA (20 ng/ml)
were then added for 60 min at 37 °C. Adherent cells were stained
with crystal violet, and absorbances were measured as described in the
legend to Fig. 2. Duplicate determinations were made for each data
point. The data are presented as percent of maximal adherence,
calculated as described in the legend to Fig. 3. In C, cells
were incubated for 20 min in RPMI 1640 alone (A), in RPMI
1640 plus 100 nM wortmannin (B), or in RPMI 1640 + 16 µM LY294002. Cells were then washed and stained for
CD11a and CD18 as described in the legend to Fig. 1. Results are
expressed as histograms of fluorescence intensity and MFI indices as
described in the legend to Fig. 1. In A and B the
values shown are the averages of two independent determinations
obtained in separate experiments. C shows results obtained
in one of two independent experiments that yielded similar
results.
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PI 3-kinase inhibitors have been shown to affect the expression of some
cell surface receptors (34). Experiments were done, therefore, to
examine the effects of wortmannin and LY294002 on the expression of
CD11a and CD18. The data shown in Fig. 4C indicate that high
concentration of inhibitors (100 nM wortmannin and 16 µM LY294002) resulted in only small changes (20-30%
reductions) in surface expression of CD18 and CD11a. These modest
changes appear insufficient to explain the marked attenuation of
LFA-1-mediated adherence to sICAM-1. Binding of LPS to cells was also
examined using FITC-LPS and FACS analysis. LPS binding was not altered in THP-1wt cells pretreated with either wortmannin or LY294002 (data
not shown).
The requirement for PI 3-kinase in LPS-induced adherence was also
examined in cells transfected with a dominant negative mutant of p85
(
p85). Stable transfection with
p85 resulted in a significant reduction in LPS-stimulated PI 3-kinase activity (Fig.
5A), and this correlated with
marked attenuation of cell adherence to sICAM-1 (Fig. 5B).
In contrast, cells transfected with wild-type p85 showed both
LPS-stimulated PI 3-kinase activity and adherence. Taken together,
these findings suggest that PI 3-kinase activation plays a central role
in LPS-induced adherence.

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Fig. 5.
PI 3-kinase activity and adherence of U937
cells transfected with either wild-type bovine p85 or dominant
negative mutant p85 . A, cells were stimulated
with either 1 µg/ml LPS or medium alone followed by detergent lysis
and immunoprecipitation with anti-PI 3-kinase antibody.
Phosphatidylinositol kinase activity was assayed as described under
"Experimental Procedures." Radioactivity observed at the origin
(ORI) reflects residual, water-soluble
32P-labeled material in the samples. Spots corresponding to
phosphatidylinositol phosphate (PIP) were cut and analyzed
by scintillation counting. Activities, expressed as percent of control
(untreated) cells transfected with wild-type p85 , are shown at the
top of the figure. B, 1 × 105
cells of either p85 or p85 transfected U937 cells were exposed
to LPS (indicated concentration) in the presence of 0.5%
AB+ and allowed to adhere to either BSA or sICAM-1 for 60 min at 37 °C and 5% CO2. Unbound cells were washed
away, and adherence was assayed as described in the legend to Fig. 2.
The data shown in A are the means ± S.E. of values
obtained in three separate experiments. Results in B are
from one of two independent experiments that yielded similar
results.
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Rho Regulates PI 3-Kinase Activation and LPS-induced
Adherence--
Small GTP-binding proteins of the Rho family
participate in various important signaling pathways, including those
regulating cellular adhesion (35-37). C. difficile toxin B,
which specifically inhibits Rho proteins (38, 39), was used to
investigate the potential role of Rho in the regulation of LPS-induced
adherence. Pretreatment for 30 min with 2 nM toxin B
resulted in significant and maximal attenuation of LPS-induced
adherence of THP-1wt to sICAM-1 (Fig.
6A) (63.5 ± 5.0%
inhibition, mean ± S.E., n = 3). In contrast,
induction of adherence in response to PMA was toxin-resistant. To
analyze further the inhibitory effect of toxin B, a dose response analysis was performed. Three separate experiments showed that IC50 for toxin inhibition of LPS-induced adherence was ~4
nM and that 20 nM produced maximal inhibition
(86.0 ± 2.8% inhibition, mean ± S.E., n = 3). In contrast, maximal inhibition of PMA-induced adherence was less
than 20%. To address whether Rho regulates activation of PI 3-kinase
in response to LPS, cells were incubated with toxin B under the same
experimental condition as those used for adherence and then stimulated
with LPS for 20 min. The data shown in Fig. 6C indicate that
toxin B reduced LPS-induced PI 3-kinase activity in a
dose-dependent manner.

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Fig. 6.
C. difficile toxin B attenuates both
LPS-induced adherence and PI 3-kinase activation. 1 × 105 THP-1wt cells were dispensed into sICAM-1-coated
microtiter wells and incubated at 37 °C and 5% CO2 with
2 nM of toxin for the times indicated (A) or for
30 min with various toxin concentrations (B). LPS (1 µg/ml
in 0.5% AB+ serum) or PMA (20 ng/ml) were then directly
added to the wells for 30 min at 37 °C. Adherent cells were assayed
as described in the legend to Fig. 2, and data are presented as percent
of maximal adherence, calculated as described in the legend to Fig. 3.
In C, cells pretreated with toxin B (indicated
concentrations for 30 min) were stimulated with 1 µg/ml LPS in 0.5%
AB+ serum or medium alone for 20 min followed by PI
3-kinase assay as described under "Experimental Procedures." The
upper rectangle shows phosphatidylinositol phosphate
(PIP) spots, and the graph below shows the corresponding
activities calculated as described in the legend to Fig. 5B.
In A and B the values shown are the averages of
two independent determinations obtained in separate experiments. The PI
3-kinase activities shown in C are the means ± S.E. of
values obtained in three separate experiments. The autoradiograph shown
in the upper portion of C is from one of the
latter three experiments and is representative of results obtained in
the two companion experiments.
|
|
LPS-induced Adherence Is
Cytohesin-1-dependent--
Cytohesin-1 is an adaptor molecule
that interacts specifically with the cytoplasmic tail of CD18 to
increase cell adhesion to ICAM-1 (11). It is also known that binding of
PtdIns-3,4,5-P3 to the pleckstrin homology domain of
cytohesin-1 is required for activating cellular adhesion (12). Given
that cytohesin-1 is not ubiquitously expressed (11), expression of this
adaptor molecule in THP-1 cells was examined. Semiquantitative RT-PCR using primers for cytohesin-1 was carried out using total RNA of THP-1
cells. The results shown in Fig. 7
demonstrate that cytohesin-1 mRNA is expressed in both THP-1wt and
THP-1rsv cells. An antisense strategy to inhibit cytohesin-1 expression
was used to examine whether cytohesin-1 is involved in LPS-induced
adherence. THP-1wt cells were incubated in the presence of antisense
S-oligos spanning the cytohesin-1 translation initiation region
(including the ATG initiation codon) and then assayed for adherence in
response to LPS. As shown in Fig. 8A,
treatment of cells with antisense S-oligo to cytohesin-1 mRNA
significantly attentuated LPS-induced adherence in a
concentration-dependent manner with maximal inhibition (72 ± 8%) at 5 µM. In contrast, at 5 µM of
control, sense S-oligo, only a minimal effect on adherence was
observed. This finding suggested a direct role of cytohesin-1 in the
response of THP-1 cells to LPS. FACS analysis of cells exposed to
fluorescein modified antisense S-oligo, in the same conditions used for
unmodified S-oligos, revealed that THP-1 cells readily incorporated
foreign DNA. However, a significant proportion of cells (24%) remained S-oligo-free (Fig. 8B). This finding may explain why
antisense S-oligo treatment did not result in complete inhibition of
LPS-induced adherence. Increasing the concentration of either S-oligos,
LipofectAMINE, or both, to achieve a transfection rate approaching
100% resulted in toxicity thereby reducing the specificity of the
antisense S-oligo treatment (data not shown).

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Fig. 7.
Expression of cytohesin-1 mRNA in
THP-1rsv and THP-1wt cells. Total RNA from exponentially growing
cells was extracted, and RT-PCR was carried out for cytohesin-1 and
actin as described under "Experimental Procedures." Lane
A, THP-1wt; lane B, THP-1rsv. Negative controls
consisting of no DNA and RNA without reverse transcriptase were
included, and no signals were obtained (data not shown). Serial
dilutions of input cDNA were examined to ensure that the
amplification was in the linear range for the PCR reaction.
|
|

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Fig. 8.
Cytohesin-1 antisense S-oligos
inhibit LPS-induced adherence to ICAM-1. A, THP-1wt
cells were incubated with various concentrations of S-oligos for 2 h at 37 °C in 250 µl of RPMI 1640 containing 2.5% LipofectAMINE.
The medium was then adjusted to 1 ml and supplemented with 10% FCS,
and culture was continued for 18 h. Cells were then washed and
tested in the adhesion assay as described in the legend to Fig. 2.
B, THP-1wt cells were incubated with 5 µM
fluoresceinated-5' antisense S-oligo using the same conditions as for
unmodified S-oligo. Cells were washed and analyzed by FACS as described
in the legend to Fig. 1. The data shown in A are the
means ± S.E. of values obtained in three independent experiments.
Results in B represent one of two independent experiments
with similar results. The histogram on the left in
B represents cells incubated with medium alone
(autofluorescence), and the histogram on the right
corresponds to cells incubated with fluorescein-labeled, sense
S-oligo.
|
|
 |
DISCUSSION |
This study examined signaling events required for LPS-induced
adherence. The system used involved a quantitative, microtiter adhesion
assay, CD14 transfected THP1 cells, and immobilized sICAM-1. Adherence
in this system was found to be dependent upon CD14 (Fig. 2).
Experiments that examined competitive inhibition of LPS-induced adherence using mAbs to CD14, CD18, and CD11a (Fig. 3A)
provided direct evidence that LPS-induced adherence to sICAM-1 involves a CD14 mediated signal leading to activation of cell surface expressed LFA-1. These findings are consistent with previous data showing that
antibody cross-linking of cell surface CD14 induces LFA-1 activation
(40). LPS effects on LFA-1 did not involve changes in the expression of
CD18 or CD11a. This indicates that LPS-induced adhesion was related to
increased affinity of LFA-1 for ICAM-1 rather than to increased
expression of cell surface LFA-1. Such changes in the properties of
LFA-1 are presumably mediated by a specific pathway of inside-out
signaling initiated through CD14.
The requirement for PI 3-kinase activity in a variety of leukocyte
functions, together with its apparent role in the adhesion of platelets
(41), lymphocytes (42), and neutrophils (33), made this enzyme an
attractive candidate for mediating signaling through CD14 for monocyte
adhesion. This hypothesis was supported further by the finding that LPS
induces the CD14-dependent association of an activated form
PI 3-kinase with p53/p56lyn (20). The role of PI 3-kinase in
LPS-induced adherence was examined using two different approaches. The
first involved the use of two structurally unrelated PI 3-kinase
inhibitors wortmannin and LY294002. LPS-induced adherence was
attenuated by both of these agents (Fig. 4). The effects of wortmannin
are considered to be relatively specific for PI 3-kinase at
concentrations similar to those used in this study (50 nM,
Fig. 4). However, the compound has been shown to inhibit phospholipase
A2 with an IC50 similar to that reported
previously for PI 3-kinase (43). On the other hand, the structurally
unrelated compound, LY294002, has been shown to inhibit PI 3-kinase by
a distinct mechanism (44). Moreover, LY294002 shows no inhibitory
effects on other lipid kinases or on several protein kinases, including
protein kinase C and mitogen-activated protein kinase (44). The
findings that both compounds inhibited LPS-induced adherence,
therefore, support the argument that PI 3-kinase is involved in the
regulation of adherence in response to LPS. This conclusion is
supported further by experiments in which a dominant negative mutant of
PI 3-kinase (
p85) expressed in U937 cells completely abrogated
LPS-induced adherence to sICAM-1 (Fig. 5B). It has been
shown previously that incubation of monocytes with LPS activates PI
3-kinase, leading to increased cellular levels of
PtdIns-3,4,5-P3 (20). Thus, the most likely mechanism for
the attenuation of adherence by either wortmannin, LY294002, or
p85
is inhibition of the formation of PtdIns-3,4,5-P3.
It has been reported previously that the small G-protein Rho regulates
PI 3-kinase activation in different cell systems (45-47). This finding
suggested the possibility that LPS-induced adherence may be
Rho-regulated and mediated by PI 3-kinase. In this report, a
requirement for Rho in LPS-induced adherence was suggested by studies
that used C. difficile toxin B, which specifically inhibits Rho family proteins (38, 48). Pretreatment of THP-1wt cells with toxin
B for 30 min. attenuated LPS-induced adherence to sICAM-1 (Fig. 6,
A and B). In contrast, PMA-induced adherence
appeared to be mediated by a toxin B-insensitive pathway (Fig. 6,
A and B). This dichotomy is consistent with
reports showing that PMA-induced responses in a variety of cell types
may be either resistant to Rho toxins or that inhibition of these
responses requires prolonged periods of incubation with toxins (24 h
and more) (49, 50). For example 8-24-h of pretreatment with botulinum
C3 exoenzyme, another inhibitor of Rho family proteins, was required to
attenuate PMA-induced, LFA-1/ICAM-1-dependent aggregation
of the lymphoblastoid cell line JY (49). On the other hand, PMA-induced
activation of phospholipase D in HEK-98 (human embryonic kidney) cells
was resistant to treatment with C. difficile toxin B for as
long as 24 h (50). In the present study, THP-1 cells were
incubated with toxin B for up to a maximum of 3 h. Under these
conditions it is clear that adherence induced by LPS was
toxin-sensitive, whereas the response to PMA was markedly resistant.
The findings that both Rho and PI 3-kinase appeared to be essential for
LPS-induced adherence raised the question as to whether they act
independently or whether they are positioned together in a single
signaling pathway. Fig. 6C shows that toxin B prevented activation of PI 3-kinase in LPS-stimulated THP-1wt cells, suggesting that Rho regulates this LPS response in monocytes. This observation is
consistent with previous reports showing involvement of Rho in PI
3-kinase activation in other systems (45-47). Although we cannot
completely eliminate the possibility of a direct, PI
3-kinase-independent role for Rho in regulating monocyte adherence, the
data suggest that LPS triggers Rho-mediated activation of PI 3-kinase,
leading to downstream effects on LFA-1 and monocyte adherence.
An important question arising from these observations is how PI
3-kinase activation modulates the properties of LFA-1. Recently, cytohesin-1 has been shown to interact with the cytoplasmic tail of
CD18 (11). Cytohesin-1 contains a domain homologous to the yeast Sec7
gene product and a pleckstrin homology domain. The Sec7 domain binds to
and regulates LFA-1 (11), and this process is positively regulated by
the binding of the PI 3-kinase metabolite PtdIns-3,4,5-P3
to the cytohesin-1 pleckstrin homology domain (12). Because a suitable
antibody to cytohesin-1 was not available, RT-PCR of THP-1 mRNA was
used to examine whether cytohesin-1 is expressed in THP-1 cells. The
results shown in Fig. 7 confirmed that the cytohesin-1 gene is
transcribed under basal conditions. To directly address the role of
cytohesin-1 in LPS-induced adherence, cytohesin-1-specific antisense
oligonucleotides were used. To ensure maximal specificity of the
antisense oligonucleotide, the sequence selected was from a region
lacking significant homology with other sequenced human genes. The
oligonucleotides were also phosphorothioate-modified to limit
degradation and purified by high performance liquid chromatography to
remove incomplete synthesis products. In addition fluorescein-modified
antisense and FACS analysis were used to monitor oligonucleotide
incorporation into cells. The finding that antisense treatment of THP-1
cells, but not treatment with sense oligonucleotide, significantly
attenuated LPS-induced adherence to ICAM-1 (Fig. 8A)
provided compelling evidence to suggest that cytohesin-1 plays an
essential role in adherence induced by LPS. Of note, the proportion of
cells that incorporated antisense-oligonucleotide (Fig. 8B)
correlated closely with the fraction of cells that failed to adhere in
response to LPS (Fig. 8A).
Taken together, the results presented are consistent with a model (Fig.
9) in which LPS binding to CD14 switches
on the small G-protein Rho leading to activation of PI 3-kinase. This
leads to increased monocyte adherence, dependent upon changes in the adhesive properties of LFA-1. The latter appears to involve the interaction of PtdIns-3,4,5-P3, cytohesin-1, and LFA-1.

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Fig. 9.
LPS-induced, monocyte adherence to
ICAM-1. Monocyte adhesion to ICAM-1 involves activated LFA-1 and
the adaptor molecule cytohesin-1. The latter is believed to be
dependent on PtdIns-3,4,5-P3 (PIP3). LPS binding
to CD14 (1) engages the small G protein Rho (2) leading to
activation of PI 3-kinase (3). PtdIns-3,4,5-P3
(4) binds to the pleckstrin homology (PH) domain
of cytohesin-1, thereby modifying its interaction, through its Sec7
domain, with the cytoplasmic tail of CD18 (5). This leads to
altered properties of LFA-1 (6) and to increased affinity
for its counter receptor ICAM-1.
|
|
 |
ACKNOWLEDGEMENTS |
We thank Dr. J. R. Woska, Jr. for
sICAM-1, Dr. Glen Armstrong for toxin B, Dr. W. C. Van Voorhis for
antibody to CD14, and Dr. R. Ulevitch for THP-1wt and THP-1rsv cells.
 |
FOOTNOTES |
*
This work was supported by Medical Research Council of
Canada Grant MT-8633.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Division of
Infectious Diseases, University of British Columbia, Rm. 452D, 2733 Heather St., Vancouver, British Columbia V5Z 3J5, Canada. Tel.: 604-875-4011; Fax: 604-875-4013; E-mail: ethan{at}unixg.ubc.ca.
The abbreviations used are:
LFA-1, leukocyte
function-associated antigen-1; LPS, lipopolysaccharide; ICAM-1, intercellular adhesion molecule-1; sICAM-1, purified soluble,
recombinant ICAM-1; THP-1wt, THP-1 cells transfected with
CD14-cDNA; THP-1rsv, THP-1 cells transfected with vector alone; PI
3-kinase, phosphatidylinositol 3-kinase; PMA, phorbol 12-myristate
13-acetate; PtdIns-3, 4,5-P3, phosphatidylinositol
3,4,5-trisphosphate; HBSS, Hanks' balanced salt solution; Wp85
, wild-type bovine PI 3-kinase subunit p85;
p85
,
-chain, mutant
bovine p85
; MFI, mean fluorescence intensity; FCS, fetal calf serum; S-oligo, phosphorothioate modified oligonucleotide; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; BSA, bovine serum
albumin; RT-PCR, reverse transcription-polymerase chain reaction; FACS, fluorescence-activated cell sorter.
 |
REFERENCES |
-
Bevilacqua, M. P.,
Nelson, R. M.,
Mannori, G.,
and Cecconi, O.
(1994)
Annu. Rev. Med.
45,
361-378[CrossRef][Medline]
[Order article via Infotrieve]
-
Bevilacqua, M. P.,
Pober, J. S.,
Wheeler, M. E.,
Cotran, R. S.,
and Gimbrone, M. A., Jr.
(1985)
J. Clin. Invest.
76,
2003-2011[Medline]
[Order article via Infotrieve]
-
Wallis, W. J.,
Beatty, P. G.,
Ochs, H. D.,
and Harlan, J. M.
(1985)
J. Immunol.
135,
2323-2330[Abstract/Free Full Text]
-
Dustin, M. L.,
and Springer, T. A.
(1989)
Nature
341,
619-623[CrossRef][Medline]
[Order article via Infotrieve]
-
Van Kooyk, Y.,
van de Wiel-van Kemenade, P.,
Weder, P.,
Kuijpers, T. W.,
and Figdor, C. G.
(1989)
Nature
342,
811-813[CrossRef][Medline]
[Order article via Infotrieve]
-
Springer, T. A.
(1990)
Nature
346,
425-434[CrossRef][Medline]
[Order article via Infotrieve]
-
Lub, M.,
Van Kooyk, Y.,
and Figdor, C. G.
(1995)
Immunol. Today
16,
479-483[CrossRef][Medline]
[Order article via Infotrieve]
-
Staunton, D. E.,
Marlin, S. D.,
Stratowa, C.,
Dustin, M. L.,
and Springer, T. A.
(1988)
Cell
52,
925-933[Medline]
[Order article via Infotrieve]
-
Lollo, B. A.,
Chan, K. W. H.,
Hanson, E. M.,
Moy, V. T.,
and Brian, A. A.
(1993)
J. Biol. Chem.
268,
21693-21700[Abstract/Free Full Text]
-
Dustin, M. L.,
and Springer, T. A.
(1991)
Annu. Rev. Immunol.
9,
27-66[CrossRef][Medline]
[Order article via Infotrieve]
-
Kolanus, W.,
Nagel, W.,
Schiller, B.,
Zeitlmann, L.,
Godar, S.,
Stockinger, H.,
and Seed, B.
(1996)
Cell
86,
233-242[Medline]
[Order article via Infotrieve]
-
Klarlund, J. K.,
Guilherme, A.,
Holik, J. J.,
Virbasius, J. V.,
Chawla, A.,
and Czech, M. P.
(1997)
Science
275,
1927-1930[Abstract/Free Full Text]
-
Haslett, C.,
Worthen, G. S.,
Giclas, P. C.,
Morrison, D. C.,
Henson, J. E.,
and Henson, P. M.
(1997)
Am. Rev. Respir. Dis.
136,
9-18
-
Pohlman, T. H.,
Stanness, K. A.,
Beatty, P. G.,
Ochs, H. D.,
and Harlan, J. M.
(1986)
J. Immunol.
136,
4548-4553[Abstract/Free Full Text]
-
Smedly, L. A.,
Tonnesen, M. G.,
Sandhaus, R. A.,
Haslett, C.,
Guthrie, L. A.,
Johnston, R. B., Jr.,
Henson, P. M.,
and Worthen, G. S.
(1986)
J. Clin. Invest.
77,
1233-1243[Medline]
[Order article via Infotrieve]
-
Owen, C. A.,
Campbell, E. J.,
and Stockley, R. A.
(1992)
J. Leukocyte Biol.
51,
400-408[Abstract]
-
Schumann, R. R.,
Leong, S. R.,
Flaggs, G. W.,
Gray, P. W.,
Wright, S. D.,
Mathison, J. C.,
Tobias, P. S.,
and Ulevitch, R. J.
(1990)
Science
249,
1429-1431[Medline]
[Order article via Infotrieve]
-
Wright, S. D.,
Ramos, R. A.,
Tobias, P. S.,
Ulevitch, R. J.,
and Mathison, J. C.
(1990)
Science
249,
1431-1433[Medline]
[Order article via Infotrieve]
-
Stefanová, I.,
Corcoran, M. L.,
Horak, E. M.,
Wahl, L. M.,
Bolen, J. B.,
and Horak, I. D.
(1993)
J. Biol. Chem.
268,
20725-20728[Abstract/Free Full Text]
-
Herrera-Velit, P.,
and Reiner, N. E.
(1996)
J. Immunol.
156,
1157-1165[Abstract]
-
Herrera-Velit, P.,
Knutson, K. L.,
and Reiner, N. E.
(1997)
J. Biol. Chem.
272,
16445-16452[Abstract/Free Full Text]
-
Ward, S. G.,
June, C. H.,
and Olive, D.
(1996)
Immunol. Today
17,
187-197[CrossRef][Medline]
[Order article via Infotrieve]
-
Shimizu, Y.,
and Hunt, S. W., III
(1996)
Immunol. Today
17,
565-573[CrossRef][Medline]
[Order article via Infotrieve]
-
Hmama, Z.,
Mey, A.,
Normier, G.,
Binz, H.,
and Revillard, J. P.
(1994)
Infect. Immun.
62,
1520-1527[Abstract]
-
Hmama, Z.,
Normier, G.,
Kouassi, E.,
Flacher, M.,
Binz, H.,
and Revillard, J. P.
(1992)
Immunobiology
186,
183-198[Medline]
[Order article via Infotrieve]
-
Hara, K.,
Yonezawa, K.,
Sakaue, H.,
Ando, A.,
Kotani, K.,
Kitamura, T.,
Kitamura, Y.,
Ueda, H.,
Stephens, L.,
Jackson, T. R.,
Hawkins, P. T.,
Dhand, R.,
Clark, A. E.,
Holman, G. D.,
Waterfield, M. D.,
and Kasuga, M.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
7415-7419[Abstract]
-
Nandan, D.,
and Reiner, N. E.
(1997)
J. Immunol
158,
1095-1101[Abstract]
-
Liu, L.,
and Pohajdak, B.
(1992)
Biochim. Biophys. Acta
1132,
75-78[Medline]
[Order article via Infotrieve]
-
Liu, M. K.,
Herrera-Velit, P.,
Brownsey, R. W.,
and Reiner, N. E.
(1994)
J. Immunol.
153,
2642-2652[Abstract/Free Full Text]
-
Sanchez-Madrid, F.,
Krensky, A. M.,
Ware, C. F.,
Robbins, E.,
Strominger, J. L.,
Burakoff, S. J.,
and Springer, T. A.
(1982)
Proc. Natl. Acad. Sci. U. S. A.
79,
7489-7493[Abstract]
-
Smith, C. W.,
Rothlein, R.,
Hughes, B. J.,
Mariscalco, M. M.,
Rudloff, H. E.,
Schmalstieg, F. C.,
Anderson, D. C.,
and Speros, P.
(1988)
J. Clin. Invest.
82,
1746-1756[Medline]
[Order article via Infotrieve]
-
Philips, M. R.,
Buyon, J. P.,
Winchester, R.,
and Weissmann, G.
(1988)
J. Clin. Invest.
82,
495-501[Medline]
[Order article via Infotrieve]
-
Capodici, C.,
Hanft, S.,
Feoktistov, M.,
and Pillinger, M. H.
(1998)
J. Immunol.
160,
1901-1909[Abstract/Free Full Text]
-
Kundra, R.,
and Kornfeld, S.
(1998)
J. Biol. Chem.
273,
3848-3853[Abstract/Free Full Text]
-
Ridley, A. J.,
Paterson, H. F.,
Johnston, C. L.,
Diekmann, D.,
and Hall, A.
(1992)
Cell
70,
401-410[Medline]
[Order article via Infotrieve]
-
Laudanna, C.,
Campbell, J. J.,
and Butcher, E. C.
(1996)
Science
271,
981-983[Abstract]
-
Laudanna, C.,
Campbell, J. J.,
and Butcher, E. C.
(1997)
J. Biol. Chem.
272,
24141-24144[Abstract/Free Full Text]
-
Just, I.,
Fritz, G.,
Aktories, K.,
Giry, M.,
Popoff, M. R.,
Boquet, P.,
Hegenbarth, S.,
and von Eichel-Streiber, C.
(1994)
J. Biol. Chem.
269,
10706-10712[Abstract/Free Full Text]
-
Just, I.,
Selzer, J.,
Wilm, M.,
von Eichel-Streiber, C.,
Mann, M.,
and Aktories, K.
(1995)
Nature
375,
500-503[CrossRef][Medline]
[Order article via Infotrieve]
-
Beekhuizen, H.,
Blokland, I.,
and Van Furth, R.
(1993)
J. Immunol.
150,
950-959[Abstract/Free Full Text]
-
Toker, A.,
Bachelot, C.,
Chen, C. S.,
Falck, J. R.,
Hartwig, J. H.,
Cantley, L. C.,
and Kovacsovics, T. J.
(1995)
J. Biol. Chem.
270,
29525-29531[Abstract/Free Full Text]
-
Shimizu, Y.,
Mobley, J. L.,
Finkelstein, L. D.,
and Chan, A. S.
(1995)
J. Cell Biol.
131,
1867-1880[Abstract]
-
Cross, M. J.,
Stewart, A.,
Hodgkin, M. N.,
Kerr, D. J.,
and Wakelam, M. J. O.
(1995)
J. Biol. Chem.
270,
25352-25355[Abstract/Free Full Text]
-
Vlahos, C. J.,
Matter, W. F.,
Hui, K. Y.,
and Brown, R. F.
(1994)
J. Biol. Chem.
269,
5241-5248[Abstract/Free Full Text]
-
Zhang, J.,
King, W. G.,
Dillon, S.,
Hall, A.,
Feig, L.,
and Rittenhouse, S. E.
(1993)
J. Biol. Chem.
268,
22251-22254[Abstract/Free Full Text]
-
Gómez, J.,
García, A.,
Borlado, L. R.,
Bonay, P.,
Martínez-A, C.,
Silva, A.,
Fresno, M.,
Carrera, A. C.,
von Eichel-Streiber, C.,
and Rebollo, A.
(1997)
J. Immunol.
158,
1516-1522[Abstract]
-
Zheng, Y.,
Bagrodia, S.,
and Cerione, R. A.
(1994)
J. Biol. Chem.
269,
18727-18730[Abstract/Free Full Text]
-
Just, I.,
Richter, H. P.,
Prepens, U.,
von Eichel-Streiber, C.,
and Aktories, K.
(1994)
J. Cell Sci.
107,
1653-1659[Abstract/Free Full Text]
-
Tominaga, T.,
Sugie, K.,
Hirata, M.,
Morii, N.,
Fukata, J.,
Uchida, A.,
Imura, H.,
and Narumiya, S.
(1993)
J. Cell Biol.
120,
1529-1537[Abstract]
-
Schmidt, M.,
Voß, M.,
Thiel, M.,
Bauers, B.,
Grannaß, A.,
Tapp, E.,
Cool, R. H.,
de Gunzburg, J.,
von Eichel-Streiber, C.,
and Jakobs, K. H.
(1998)
J. Biol. Chem.
273,
7413-7422[Abstract/Free Full Text]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.