Department of Chemical Engineering, Ohio University, Athens, Ohio 45701
Submitted 17 March 2003 ; accepted in final form 2 June 2003
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ABSTRACT |
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endothelial cell adhesion molecules; inflammation; cytokines; proteasome inhibitor
The cytokine-induced expression of these ECAMs is regulated at the gene
level by the activity of transcription factors. For example, the promoter
regions of genes encoding for E-selectin, VCAM-1, and ICAM-1 all have binding
sites for the transcription factor nuclear factor-B (NF-
B)
(19,
24,
32,
39). NF-
B is present in
the cytoplasm of unstimulated endothelial cells and is rendered inactive due
to its association with the inhibitory protein I
B
(21). IL-1
or
TNF-
stimulation of endothelial cells induces the phosphorylation of
I
B (21). The
phosphorylated form of I
B is then ubiquitinated and degraded via the
proteasome-dependent pathway, eventually leading to its dissociation from
NF-
B (21). The
resulting nuclear translocation of the active NF-
B leads to
transcription, translation, and expression of a large number of
NF-
B-dependent genes, which include the genes encoding for E-selectin,
VCAM-1, and ICAM-1 (21).
Several current or potential therapeutics for pathological inflammation
work, at least in part, by modulating the activity of transcription factors
(8,
26,
27,
29,
34,
38). For example, proteasome
inhibitors can suppress the TNF--induced expression of ECAMs by
inhibiting the degradation of phosphorylated I
B and thus blocking
NF-
B activation (1,
17,
29). The inhibition of ECAM
expression contributes to a reduction in leukocyte adhesion and transmigration
across the endothelium (1,
17,
29). Although a variety of in
vitro studies have used transcription inhibitors to elucidate the mechanism of
ECAM gene regulation, the scope of these studies, in terms of the use of the
inhibitors as anti-inflammatory agents, is limited. Specifically, the majority
of the studies have focused on the action of an inhibitor given to the
endothelial cells exposed to a single proinflammatory cytokine (in nearly all
cases, TNF-
) for a relatively short period of time (i.e., <24 h with
the primary focus on 4-6 h) (1,
17,
26,
29,
38). As therapeutic in the in
vivo setting, the transcription inhibitors will need to act on endothelial
cells in an environment with more than one cytokine and on endothelial cells
that are exposed to cytokines for extended periods of time. Additionally,
although certain cytokines (e.g., IL-1
and TNF-
) have somewhat
similar effects on the endothelium, their effects do not appear to be
identical. In particular, it has been observed that HUVEC that have become
refractory to IL-1-induced expression of E-selectin can be induced to express
E-selectin by TNF-
(28).
The above considerations led us to further probe the effect of a
transcription inhibitor, the proteasome inhibitor lactacystin
(17), on IL-1 and
TNF-
induction of ECAMs and consequent myeloid-endothelial cell
adhesion. Specifically, we focused on determining whether lactacystin can
inhibit ECAM expression and myeloid cell adhesion to 1) endothelial
cells activated by concurrent treatment with IL-1
and TNF-
,
2) endothelial cells activated by sequential treatment with
IL-1
and TNF-
(i.e., heterocytokine stimulation of endothelial
cells that have become refractory to homocytokine stimulation), and
3) endothelial cells exposed to TNF-
for a relatively long
time period (i.e., 24 h).
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MATERIALS AND METHODS |
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Antibodies. Function-blocking murine MAb HEL3/2 (anti-human E-selectin; IgG1) was a generous gift from Dr. Raymond T. Camphausen (Wyeth Laboratories, Cambridge, MA). Function-blocking murine MAb 51-10C9 (anti-human VCAM-1; IgG1) and murine MAb G46-2.6 (anti-human MHC class-I antigens; IgG1) were obtained from BD Pharmingen (San Diego, CA). Murine MAb R6.5 (anti-human ICAM-1; IgG2a) was kindly provided by Dr. Robert Rothlein (Boehringer Ingelheim, Ridgefield, CT). Murine MAb 15.2 (anti-human ICAM-1; IgG1) was obtained from Ancell (Bayport, MN). Murine MAb TS1/22 (anti-human LFA-1; IgG1) was obtained from Endogen (Woburn, MA). Horseradish peroxidase-conjugated goat F(ab')2 anti-mouse IgG polyclonal secondary antibody, used to detect the primary MAbs in the enzyme-linked immunosorbent assay (ELISA), was purchased from CalBiochem.
Cell culture. HUVEC were purchased from Clonetics (San Diego, CA) and maintained in culture as described previously (14). In brief, the HUVEC were cultured in M199 supplemented with 8% FBS, 100 µg/ml heparin, 50 µg/ml endothelial mitogen, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. This is referred to as HUVEC medium. HUVEC were subcultured on gelatin precoated 96-well tissue culture plates (Corning, Corning, NY) for ELISA, viability, and apoptosis assays, and on gelatin precoated 35-mm tissue culture dishes (Corning) for adhesion assays. All assays were performed with confluent HUVEC monolayers. HL60 cells were cultured in RPMI supplemented with 8% FBS, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. For the adhesion assays, HL60 cells were washed, resuspended to 1 x 108 cells/ml in RPMI, and held on ice (<4 h) until the time they were used in the flow adhesion assay. Before perfusion through the parallel plate flow chamber, HL60 cells were diluted to 5 x 105 cells/ml in assay buffer.
Lactacystin followed by individual and concurrent IL-1
and TNF-
treatment of HUVEC. HUVEC were pretreated
with 20 µM lactacystin or 0.05% DMSO (carrier control for lactacystin).
After a 1-h incubation, lactacystin and DMSO were removed and the HUVEC were
treated individually or concurrently with IL-1
and TNF-
for 4 h.
Postcytokine treatment, the ELISA and the adhesion assays were performed. Note
that imparting targeting (9,
36) to the delivery of
transcription inhibitors will greatly enhance the therapeutic potential of
these reagents (see latter part of DISCUSSION and Ref.
11). Thus, to simulate the
targeted delivery, we chose to treat the HUVEC with lactacystin in a pulse
fashion (1-h pretreatment followed by removal) rather than continuously. In
preliminary experiments, we determined the concentration of IL-1
or
TNF-
needed to give the maximal level of HL60 cell adhesion to HUVEC at
4 h postcytokine activation. All subsequent experiments were carried out with
IL-1
and/or TNF-
concentrations at or above the concentration
needed to achieve a maximal level of adhesion (0.25 ng/ml for IL-1
and
25 ng/ml for TNF-
).
Lactacystin followed by sequential IL-1 and
TNF-
treatment of HUVEC. HUVEC were treated with one of
the cytokines (either IL-1
or TNF-
) for 24 h. After this
incubation, the initial cytokine was removed and HUVEC were treated with a
fresh dose of either the same cytokine (homocytokine activation) or the other
cytokine (heterocytokine activation). After a 4-h incubation with the fresh
(either homo- or hetero-) cytokine, the ELISA and the adhesion assays were
performed. To test the effect of lactacystin on heterocytokine activation,
IL-1
that was used for the first activation was removed at 23 h and the
HUVEC were treated for 1 h with 20 µM lactacystin or 0.05% DMSO. After the
1-h incubation, lactacystin and DMSO were removed and the HUVEC were treated
with TNF-
for 4 h. After the 4-h TNF-
activation, the ELISA and
the adhesion assays were performed.
Lactacystin followed by long-term TNF- treatment of
HUVEC. HUVEC were pretreated with 20 µM lactacystin or 0.05% DMSO.
After a 1-h incubation, lactacystin and DMSO were removed and the HUVEC were
treated with either TNF-
or HUVEC medium alone for 24 h. After the 24-h
incubation, the HUVEC incubated in medium alone were treated with TNF-
for 4 h. Post-TNF-
treatments, viability, apoptosis, ELISA, and/or
adhesion assays were performed.
ELISA. ELISA was used to characterize the protein levels of adhesion molecules on HUVEC in a manner similar to that described previously (13). HUVEC were washed with cold HBSS+, fixed in 1% formaldehyde at 4°C for 20 min, washed with cold HBSS+, and incubated in cold M199 containing 8% FBS. Unless otherwise noted, from this point on all antibody dilutions and washes were carried out with M199 containing 8% FBS. Murine MAbs (primary MAbs) to ECAMs were added (10 µg/ml), and the HUVEC were incubated at 4°C for 20 min. After the incubation, the wells were washed and a peroxidase-conjugated polyclonal (secondary) antibody to mouse IgG was added (diluted 1:50). After a 20-min incubation at 4°C, the wells were washed and treated with OPD dissolved in phosphate citrate buffer containing sodium perborate. After a 10-min incubation at room temperature, the absorbance of the fluid in each well was determined at 450 nm using a microwell plate spectrophotometer (Molecular Devices, Sunnyvale, CA). In every experiment, each condition was run in triplicate wells.
Flow adhesion assays. A parallel plate flow chamber (Glycotech, Rockville, MD), similar to that described by McIntire, Smith, and colleagues (15), was used in this study. Our particular set up has been described previously (35). Temperature was maintained at 37°C with a heating plate. A 35-mm tissue culture dish containing a confluent HUVEC monolayer was loaded into the flow chamber. The flow chamber was mounted on an inverted microscope connected to a charge-coupled device videocamera, VCR, and monitor. After a brief rinse, HL60 cells (5 x 105 cells/ml in assay buffer) were drawn over the HUVEC monolayer at a shear stress of 1.8 dynes/cm2. To determine HL60 cell accumulation (HL60 cells/mm2 in figures), the number of HL60 cells adherent (either rolling or firmly adherent, i.e., not moving for 2 s) to the HUVEC monolayer in 8 different fields of view after 2.5 min of flow was determined, normalized to the area of the field of view, and averaged to give the result for that particular run. Such an assay was done n number of times (as indicated in the figure legends), and the values were averaged to give the results presented in the figures. In certain experiments, cytokine-activated HUVEC were pretreated with MAbs (10 µg/ml in HUVEC medium) 15 min before use in the adhesion assays.
Viability assay. A cell proliferation assay kit (Promega, Madison, WI), containing the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS tetrazolium salt), was used to assess the viability of the HUVEC. Viable cells reduced MTS to form a colored product. The protocol used was per the manufacturer's instructions. Briefly, a MTS/phenazine methosulfate (PMS) solution was added to the wells of a 96-well plate containing the HUVEC. After a 1-h incubation at 37°C, the absorbance of the fluid in each well was determined at 490 nm using the microwell plate spectrophotometer. In every experiment, each condition was run in at least six different wells.
Apoptosis assay. The Cell Death Detection ELISA Kit (Roche Applied Science, Indianapolis, IN) was used to assess apoptosis of HUVEC. The protocol followed was per the manufacturer's instructions. In brief, HUVEC monolayers were washed and treated with lysis buffer for 30 min at room temperature. The lysate was then centrifuged (200 g) for 10 min, and 20 µl of lysate supernatants were transferred to separate wells of a streptavidin-coated microplate. Subsequently, 80 µl of immunoreagent, containing a combination of anti-histone-biotin and anti-DNA-POD, was added to each well. After a 2-h incubation at room temperature, the wells were extensively washed and treated with 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) solution. After a 15-min incubation at room temperature, the absorbance of the fluid in each well was determined at 405 and 490 nm (reference reading) using the microwell plate spectrophotometer. In every experiment, each condition was run in triplicate wells.
Statistics. A single-factor analysis of variance (ANOVA) was used to assess the presence of statistical differences. If ANOVA indicated significant differences between conditions, a Bonferroni test was used for multiple pair-wise comparisons. P values <0.001 (for ELISA) and <0.05 (for adhesion, viability, and apoptosis assays) were considered statistically significant. Unless stated otherwise, all error bars represent SD.
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RESULTS |
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To investigate the functional consequence of the above results, we studied
the adhesion of HL60 cells to HUVEC under flow conditions. We chose to use
HL60 cells, as opposed to freshly isolated leukocytes, because we were
primarily interested in the effect of lactacystin on ECAM expression and
adhesion as opposed to chemokine expression and transmigration. The HL60 cells
we used exhibited little, if any, transmigration. As shown in
Fig. 1E, a significant
number of HL60 cells adhered to 4-h concurrent IL-1- and
TNF-
-activated HUVEC, whereas very few, if any, HL60 cells adhered to
unactivated HUVEC. Pretreatment of HUVEC with 20 µM lactacystin for 1 h
before 4-h concurrent activation with IL-1
and TNF-
significantly
reduced HL60 cell adhesion (Fig.
1E), whereas pretreatment with 0.05% DMSO had little, if
any, effect on HL60 cell adhesion (Fig.
1E). Combined, the results presented in this section
clearly demonstrate, for the first time, that lactacystin can significantly
reduce ECAM expression and HL60 cell adhesion to 4-h concurrent IL-1
-
and TNF-
-activated HUVEC.
Lactacystin inhibits 4-h TNF--induced expression of
E-selectin and VCAM-1 and HL60 cell adhesion to HUVEC that have become
refractory to IL-1
activation. In addition to acting
concurrently on the endothelium, IL-1
and TNF-
may work together
to sequentially activate the endothelium
(28). Thus we next probed the
effects of lactacystin on ECAM expression and HL60 cell adhesion to HUVEC
activated by sequential treatment with these two cytokines. Previous studies
have shown that HUVEC treated for 24 h with a cytokine (i.e., IL-1 or
TNF-
) become desensitized, or refractory, to reinduction of E-selectin
by a fresh dose of the same (homo) cytokine
(28). The refractory HUVEC do,
however, express E-selectin in response to reactivation by a heterocytokine
(28). We sought to determine
whether lactacystin could block the heterocytokine reactivation of HUVEC that
have become refractory to homocytokine stimulation. Note that we focused on
TNF-
activation of IL-1
-refractory HUVEC because preliminary
experiments indicated that IL-1
treatment of TNF-
-refractory
HUVEC had little effect on ECAM expression or myeloid cell adhesion (data not
shown).
The level of E-selectin expression on HUVEC treated with IL-1 for 24
h had returned (from the level at the 4-h IL-1
timepoint) to near basal
levels (Fig. 2A),
whereas the levels of VCAM-1 and ICAM-1 (relative to unactivated HUVEC)
(Fig. 2, B and
C) remained elevated. Treatment of 24-h
IL-1
-activated HUVEC with a fresh dose of IL-1
for 4 h caused a
relatively small increase, compared with 4 h of IL-1
activation alone,
in E-selectin expression (Fig.
2A) and caused no increase in VCAM-1 or ICAM-1 expression
(Fig. 2, B and
C). The HUVEC had become desensitized, or refractory, to
reactivation with IL-1
. The IL-1
-refractory HUVEC did, however,
respond significantly to activation by TNF-
. Specifically, at 4 h
post-TNF-
activation, the levels of E-selectin and VCAM-1 were both
significantly increased (relative to the 24-h IL-1
timepoint)
(Fig. 2, A and
B), whereas the level of ICAM-1 remained the same
(Fig. 2C).
Pretreatment of IL-1
-refractory HUVEC with 20 µM lactacystin for 1 h
before 4-h activation with TNF-
caused a significant reduction in
TNF-
-induced expression of E-selectin and VCAM-1
(Fig. 2, A and
B) compared with pretreatment with 0.05% DMSO, which had
no effect (Fig. 2, A and
B).
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To investigate the functional consequence of the above results, we studied
the adhesion of HL60 cells to the HUVEC. As shown in
Fig. 2D, HL60 cell
adhesion to 24-h IL-1-activated HUVEC was significantly less than the
level of HL60 cell adhesion to 4-h IL-1
-activated HUVEC and only
slightly above the level of HL60 cell adhesion to unactivated HUVEC. Treatment
of 24-h IL-1
-activated HUVEC with a fresh dose of IL-1
for 4 h had
little effect on HL60 cell adhesion relative to the 24-h IL-1
timepoint
(Fig. 2D). The HUVEC
had become refractory to IL-1
-induced HL60 cell adhesion. Treatment of
IL-1
-refractory HUVEC with TNF-
for 4 h did, however,
significantly increase the level of HL60 cell adhesion relative to the 24-h
IL-1
timepoint (Fig.
2D). Pretreatment of IL-1
-refractory HUVEC with 20
µM lactacystin for 1 h before 4-h activation with TNF-
significantly
reduced TNF-
-induced increased HL60 cell adhesion
(Fig. 2D) relative to
treatment with 0.05% DMSO, which had no effect
(Fig. 2D).
Combined, the results presented in this section clearly demonstrate, for
the first time, that lactacystin can inhibit ECAM expression and HL60 cell
adhesion to HUVEC activated by sequential treatment with IL-1 and
TNF-
. Specifically, lactacystin can inhibit 4-h TNF-
-induced
expression of E-selectin, VCAM-1, and myeloid cell adhesion to HUVEC that have
become refractory to IL-1
activation.
Lactacystin inhibits 24-h TNF--induced expression of
E-selectin and VCAM-1 but not ICAM-1 and inhibits 24-h
TNF-
-induced HL60 cell adhesion to HUVEC. Because there
have been few, if any, investigations into the effect of transcription
inhibitors on HUVEC exposed to cytokines for long periods of time (i.e., 24
h), we probed the effect of lactacystin on 24-h TNF-
-induced expression
of ECAMs and myeloid cell adhesion to HUVEC. [Note that because HL60 cells
exhibit very little adhesion to 24-h IL-1
-activated HUVEC
(Fig. 2D), we chose to
focus on 24-h TNF-
-activated HUVEC.] In preliminary experiments, we
determined the effect of 24-h TNF-
treatment, by itself or subsequent
to a 1-h pretreatment with lactacystin, on HUVEC viability and apoptosis. Note
that in these experiments and throughout this study, the lactacystin was
removed from the HUVEC before the addition of TNF-
. As shown in
Fig. 3, treatment of HUVEC for
24 h with TNF-
or pretreatment of HUVEC with lactacystin for 1 h,
followed by treatment with TNF-
for 24 h, had no significant effect on
viability (Fig. 3A) or
apoptosis (Fig. 3B)
relative to untreated HUVEC. These results are consistent with a previous
report demonstrating that pretreatment of HUVEC with a combination of
lactacystin and its aqueous derivative,
-lactone, followed by 12-h
treatment with TNF-
, has no effect on metabolic activity
(2). Thus we proceeded to
determine the effect of lactacystin on 24-h TNF-
-activated HUVEC.
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HUVEC treated with TNF- for 24 h expressed a level of E-selectin
that was slightly higher than the basal level
(Fig. 4A) and
expressed elevated levels of VCAM-1 and ICAM-1, relative to unactivated HUVEC
(Fig. 4, B and
C). Note that E-selectin expression at 24 h
post-TNF-
activation was distinctly less than the level seen at 4 h
post-TNF-
treatment (Fig.
4A vs. Fig.
2A, right-most bar). Pretreatment of HUVEC with
20 µM lactacystin for 1 h before 24-h activation with TNF-
significantly reduced the induced expression of E-selectin and VCAM-1 relative
to pretreatment with 0.05% DMSO alone (Fig.
4, A and B) but had no effect on ICAM-1
expression (Fig. 4C).
To investigate the functional consequence of the above results, we studied the
adhesion of HL60 cells to the HUVEC. The level of HL60 cell adhesion to 24-h
TNF-
-activated HUVEC was significantly higher than the level of
adhesion to unactivated HUVEC (Fig.
4D). Pretreatment of HUVEC with 20 µM lactacystin for
1 h before 24-h activation with TNF-
significantly reduced HL60 cell
adhesion relative to pretreatment with 0.05% DMSO alone, which had no effect
(Fig. 4D).
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To further probe and substantiate the above results (i.e.,
Fig. 4), we sought to determine
whether lactacystin applied transiently for 1 h could reduce ECAM expression
in response to TNF- treatment applied 24 h later. Thus we pretreated
HUVEC for 1 h with lactacystin and subsequently incubated the HUVEC in HUVEC
medium for 24 h (as opposed to HUVEC medium with TNF-
, as was done in
Fig. 4). After the 24-h rest
period, we treated the HUVEC with TNF-
for 4 h and subsequently
conducted an ELISA assay. As shown in Fig.
5 (left panels), lactacystin reduced 4-h TNF-
(applied 24 h postlactacystin)-induced E-selectin and VCAM-1 expression but
had little effect on ICAM-1 expression. In line with other reports
(1,
17), we confirmed
(Fig. 5, right panels)
that 1-h pretreatment with lactacystin immediately before 4-h TNF-
activation (as opposed to having a 24-h rest period before application of
TNF-
, as was done in Fig.
5, left panels) inhibited E-selectin, VCAM-1, and ICAM-1
expression.
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Combined, the results presented in this section clearly demonstrate, for
the first time, that lactacystin inhibits E-selectin, VCAM-1 expression, and
HL60 cell adhesion to HUVEC exposed to TNF- for a relatively long
period (i.e., 24 h) but does not have an effect on ICAM-1. These results are
in contrast to lactacystin's effect on 4-h TNF-
activation of HUVEC
immediately after treatment with lactacystin, where it is observed that
lactacystin reduces expression of E-selectin and VCAM-1, as well as ICAM-1
(Refs. 1 and
17, and
Fig. 5, right
panels).
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DISCUSSION |
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To our knowledge, there have been few, if any, investigations into the
effect of lactacystin on concurrent or heterocytokine stimulation of
endothelial cells. Our work clearly demonstrates that lactacystin can
significantly reduce 4-h concurrent IL-1- and TNF-
-induced
E-selectin, VCAM-1, and ICAM-1 expression
(Fig. 1, A-C)
and myeloid cell adhesion to HUVEC under physiological levels of fluid shear
(Fig. 1E). These
findings complement previous studies that have revealed that lactacystin can
inhibit 4-h TNF-
-induced ECAM expression and leukocyte adhesion
(1,
17). The data shown in
Fig. 2 demonstrate that
although 24-h IL-1
-activated HUVEC are desensitized to reactivation by
IL-1
(i.e., IL-1
-refractory HUVEC), they do respond to
reactivation by TNF-
(heterocytokine activation), a result consistent
with other reports (28).
Clearly, lactacystin can inhibit 4-h TNF-
-induced expression of
E-selectin and VCAM-1 (Fig. 2, A
and B) and myeloid cell adhesion to IL-1
-refractory
HUVEC (Fig. 2D). These
results reveal a role for the proteasome and suggest involvement of
NF-
B in TNF-
stimulation of IL-1
-refractory HUVEC.
Interestingly, we found that treatment of 24-h TNF-
-refractory HUVEC
with IL-1
for 4 h elicited very little, if any, increase in E-selectin,
VCAM-1, and ICAM-1 expression and myeloid cell adhesion (data not shown). The
differences between this result and that reported previously
(28) may be explained, in
part, by the differences in concentrations of IL-1
and TNF-
used
in the assays.
Before the present study, there has been little, if any, investigation into
the effects of transcription inhibitors on HUVEC exposed to cytokines for long
periods of time (i.e., 24 h). We found that lactacystin can significantly
reduce 24-h TNF--induced expression of E-selectin and VCAM-1
(Fig. 4, A and
B) and 24-h TNF-
-induced myeloid cell adhesion to
HUVEC (Fig. 4D).
Lactacystin did not, however, suppress 24-h TNF-
-induced ICAM-1
expression (Fig. 4C).
It appears that lactacystin applied transiently for 1 h continued to exert an
effect at the 24-h time point because E-selectin and VCAM-1 expression was
suppressed in the experiments described in Figs.
4 and
5, left panels. Thus
the lack of ICAM-1 suppression at the 24-h time point suggests that
TNF-
induction of ICAM-1 can occur independently of the proteasome and
possibly independently of NF-
B activation. In support of this, we note
that induction pathways parallel to NF-
B activation [i.e., c-Jun
(22,
30)] have been identified,
inhibition of the proteasome pathway has been shown to induce c-Jun-dependent
transcription on fibroblast cells
(23), and ICAM-1 expression
can be induced through an NF-
B-independent mechanism by overexpression
of c-Jun and c-Fos (37). Thus
although our study clearly demonstrates that a proteasome inhibitor can reduce
long-term TNF-
induction of E-selectin and VCAM-1 and myeloid cell
adhesion, it also suggests a proteasome-independent mechanism for TNF-
induction of ICAM-1 that is detectable at 24 h postactivation but not at 4 h
postactivation. This novel and rather unexpected finding highlights the fact
that the effects of transcription inhibitors on short-term exposure of
endothelial cells to cytokines such as IL-1
and TNF-
cannot
necessarily be extrapolated to what will occur for long-term exposure.
Although the expression of ECAMs clearly correlates with myeloid cell
adhesion (Figs. 1,
2, and
4), a closer inspection of the
data suggests that the relationship between lactacystin treatment and myeloid
cell adhesion might be rather complex. For example, at the 4-h timepoint,
lactacystin appears to reduce the IL-1- and TNF-
-induced
E-selectin expression by
32% (Fig.
1A) while inhibiting
82% of the HL60 cell adhesion
(Fig. 1E). In this
context, we have made several other observations. First, function-blocking MAb
experiments suggest that E-selectin and VCAM-1 (ICAM-1 was not tested) are
involved in HL60 cell adhesion to 4-h concurrent IL-1
- and
TNF-
-activated HUVEC (data not shown). Second, HL60 cells that were
adherent to 4-h concurrent IL-1
- and TNF-
-activated HUVEC were
predominantly (93 ± 1.74%; average ± SD; n = 4) firmly
adherent. In contrast, less than half (48 ± 6.22%; average ± SD;
n = 4) of the HL60 cells that were adherent to lactacystin-pretreated
4-h concurrent IL-1
- and TNF-
-activated HUVEC were firmly
adherent (i.e., the majority were rolling). Third, a significant number of
HL60 cells exhibited a transient interaction (an interaction that lasted for a
fraction of a second) with lactacystin-pretreated 4-h concurrent IL-1
-
and TNF-
-activated HUVEC (data not shown). Such interactions were not
observed when HL60 cells were perfused over unactivated HUVEC (data not
shown). Due to the transient nature of these interactions, the majority of the
transiently adhesive HL60 cells were no longer adherent to the
lactacystin-pretreated 4-h-concurrent IL-1
- and TNF-
-activated
HUVEC when the accumulation (i.e., the data in
Fig. 1E) was
measured.
Combined, the above observations suggest that the net accumulation of
myeloid cells on the HUVEC involves a multistep cascade that relies on the
interaction of the myeloid cells with E-selectin as well as another ECAM(s).
Thus a plausible explanation of the observation in
Fig. 1 is that although
lactacystin does not completely eliminate 4-h-concurrent IL-1- and
TNF-
-induced E-selectin expression
(Fig. 1A), it does
reduce induced expression of E-selectin, VCAM-1, and ICAM-1
(Fig. 1,
A-C). These reductions, in aggregate, might be
sufficient to cause a significant inhibition in the overall accumulation of
HL60 cells on 4-h-concurrent IL-1
- and TNF-
-activated HUVEC
(Fig. 1E). Although
this explanation is consistent with the data, it is quite conceivable that
lactacystin has other effects on HUVEC (e.g., effects on receptor
presentation) that may also contribute to the reduction in myeloid cell
adhesion. Further studies are needed to completely understand the relationship
between lactacystin treatment and the consequent effects on myeloid
adhesion.
Overall, the results presented in this study bode well for using
transcription inhibitors as anti-inflammatory agents to reduce aberrant
leukocyte adhesion. However, it is important to note that these inhibitors can
have other effects on endothelial cells in addition to suppression of ECAM
expression (16,
25). The nonspecific effect of
transcription inhibitors becomes even more problematic if they are
administered systemically and could thus potentially affect all cells, in
addition to the target endothelial cells. For example, NF-B is a
ubiquitous transcription factor, and genetic deletions of NF-
B subunits
have been shown to cause lethality
(3) and suppress immune
response (33). Detrimental
side effects caused by the nonspecific action of transcription inhibitors
administered systemically lead to the idea of adopting targeting approaches to
selectively deliver the transcription inhibitors to sites of inflammation. For
example, a targeted drug delivery scheme could be employed wherein
transcription inhibitors are incorporated into drug carriers that bear a
ligand for a selectively expressed ECAM
(4,
6,
7,
9,
11,
36). Ideally, once
administered, the carriers would selectively bind to endothelium within
inflamed tissue via the ligand-ECAM chemistry and not bind to other segments
of the endothelium or other tissue. In this manner, the transcription
inhibitors could be targeted to the site of pathological inflammation, thereby
reducing the unwanted side effects due to the nonspecific action of the
inhibitors administered systemically.
E-selectin is an attractive target for selective drug delivery
(4,
6,
9,
10,
36) because it appears to be
expressed at high levels on endothelium at sites of inflammation but minimally
expressed on endothelium within normal tissue
(10,
36). The present work provides
an in vitro model to test the biological effect of E-selectin-targeted
transcription inhibitors. Specifically, the results of our study clearly
indicate that treating IL-1-refractory HUVEC with TNF-
elicits a
second wave of E-selectin expression that can support myeloid cell adhesion
(Fig. 2). Thus one can test the
feasibility of the targeting approach by attempting to inhibit the
TNF-
-elicited second wave of E-selectin expression and myeloid cell
adhesion by using a transcription inhibitor-loaded drug carrier targeted to
the first wave of E-selectin elicited by the initial cytokine activation with
IL-1
.
In summary, we have shown, for the first time, that the proteasome
inhibitor lactacystin can 1) reduce 4-h-concurrent IL-1- and
TNF-
-induced expression of E-selectin, ICAM-1, VCAM-1, and HL60 cell
adhesion to HUVEC; 2) inhibit 4-h TNF-
-induced expression of
E-selectin, VCAM-1, and HL60 cell adhesion to HUVEC that have become
desensitized to IL-1
activation; 3) inhibit 24-h
TNF-
-induced expression of E-selectin and VCAM-1 but not ICAM-1; and
4) inhibit 24-h TNF-
-induced HL60 cell adhesion to HUVEC.
Combined, our results demonstrate that a proteasome inhibitor can reduce
concurrent, sequential, and long-term IL-1
- and TNF-
-induced ECAM
expression and myeloid cell adhesion.
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DISCLOSURES |
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FOOTNOTES |
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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.
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