1 Departments of Molecular and Cellular Physiology and Medicine, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932; and 2 Department of Pediatrics, University of California, San Francisco, California 94143-0748
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ABSTRACT |
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Monolayers of
cultured endothelial cells exposed to hypoxia-reoxygenation exhibit a
transcription-dependent increase in E-selectin expression and
E-selectin-dependent neutrophil-endothelial cell adhesion. The overall
objectives of this study were 1) to determine whether
ischemia-reperfusion (I/R) promotes upregulation of E-selectin in vivo; 2) if so, to define the mediators of this response;
and 3) to assess the contribution of E-selectin to I/R-induced
neutrophil recruitment. The dual-radiolabeled monoclonal antibody (MAb)
technique was used to measure E-selectin expression in the intestinal
vasculature. Ischemia was induced by complete occlusion
(30-60 min) of the superior mesenteric artery followed by
3-24 h of reperfusion. Increasing durations of ischemia
elicited progressively increasing (2- to 5-fold) levels of E-selectin
expression, with the peak response noted after 45 min of
ischemia and 5 h of reperfusion. Subsequent experiments
revealed that I/R-induced increase in E-selectin expression (at 5 h) is
significantly blunted in transgenic mice that overexpress
Cu,Zn-superoxide dismutase or by treatment of wild-type mice with
either a blocking antibody against tumor necrosis factor (TNF)- or
an inhibitor of nuclear factor-
B (NF-
B) activation (PS341).
Administration of an E-selectin-specific MAb dramatically reduced
I/R-induced recruitment of neutrophils in the intestine. These findings
suggest that superoxide and TNF-
mediate gut I/R-induced E-selectin
expression via an NF-
B-dependent mechanism; this upregulation of
E-selectin contributes significantly to I/R-induced neutrophil recruitment.
superoxide dismutase; tumor necrosis factor; neutrophils; nuclear factor-kappaB; interferon-gamma
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INTRODUCTION |
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LEUKOCYTE-ENDOTHELIAL CELL adhesion has been implicated as a key initiating step in the pathogenesis of ischemia-reperfusion (I/R) injury. As a consequence, both in vitro and in vivo models of I/R have been used to identify and characterize the specific leukocyte and endothelial cell adhesion molecules (CAMs) that contribute to I/R-induced leukocyte recruitment and tissue injury (13, 23). Monolayers of cultured endothelial cells exposed to hypoxia-reoxygenation (H/R) have proven to be particularly useful for defining the time course and magnitude of endothelial CAM expression after H/R as well as the chemical and molecular processes that underlie these changes. For example, we (17) recently reported that cultured human umbilical vein endothelial cells (HUVECs) exposed to H/R become hyperadhesive to human neutrophils and exhibit a biphasic response, with peak adhesion occurring at 30 min (phase 1) and 240 min (phase 2) after reoxygenation. Blocking monoclonal antibodies (MAbs) were used to reveal a dominant role for P-selectin and intercellular adhesion molecule 1 (ICAM-1) in mediating the phase 1 neutrophil adhesion, whereas E-selectin had a dominant role in mediating the phase 2 adhesion response. The relative contribution of these CAMs to H/R-induced neutrophil adhesion was corroborated by the kinetics of surface expression of ICAM-1 and P- and E-selectin on posthypoxic HUVECs (17).
In addition to revealing an important contribution of E-selectin to the
neutrophil-endothelial cell adhesion observed several hours after
reoxygenation, in vitro models of I/R have provided evidence that
implicates enhanced oxygen radical production as a key event that
initiates the increased transcription of E-selectin. HUVECs exposed to
H/R exhibit a profound increase in the ratio of oxidized (GSSG) to
reduced (GSH) glutathione, and pharmacological agents that produce
comparable changes in GSSG/GSH in normoxic HUVEC monolayers also elicit
a biphasic increase in neutrophil adhesion and increased endothelial
CAM expression similar to those observed in posthypoxic monolayers
(20). The increased endothelial expression of E-selectin that
accompanies the H/R- or drug-induced oxidant stress (increased
GSSG-to-GSH ratio) is greatly attenuated by inhibitors of the nuclear
transcription factor nuclear factor-B (NF-
B) (17, 20). Hence, the
results derived from posthypoxic HUVECs suggest that an oxidant stress
results in the activation of NF-
B, which in turn stimulates the
transcription-dependent expression of E-selectin that subsequently
mediates the neutrophil adhesion observed 4 h after reoxygenation.
Although the in vitro models of I/R clearly implicate a role for
E-selectin in the recruitment of neutrophils after reperfusion (reoxygenation), relatively little is known about the magnitude and
kinetics of expression of this endothelial CAM in the
postischemic microvasculature. It also remains unclear whether
the mechanisms that elicit E-selectin expression in posthypoxic HUVECs
are equally relevant to the more complex in vivo situation, in which
postischemic endothelial cells are exposed to a multitude of
inflammatory mediators that are released by a variety of auxiliary
cells (e.g., mast cells, macrophages, platelets) that are also
activated by I/R (12, 23). Hence, the overall objectives of this study
were 1) to determine whether I/R promotes the upregulation of
E-selectin in vivo; 2) if so, to define the contributions of
specific cytokines [tumor necrosis factor (TNF)-,
interferon-
], superoxide, and NF-
B to I/R-induced
E-selectin expression; and 3) to assess the importance of
E-selectin in mediating I/R-induced neutrophil recruitment.
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MATERIALS AND METHODS |
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Monoclonal antibodies The MAbs used for the in vivo characterization of E-selectin expression were 10E9.6, a rat IgG2a directed against mouse E-selectin (4), and P-23, a nonbinding murine IgG1 directed against human P-selectin (24). 10E9.6 was purchased from Pharmingen (San Diego, CA) and P-23 was provided by Dr Donald C. Anderson (Pharmacia & Upjohn, Kalamazoo, MI).
Although we showed previously (10) that MAb 10E9.6 is an excellent binding antibody to vascular endothelial cells in C57BL/6 mice, the ability of this MAb to functionally block neutrophil-endothelial cell interactions in vivo is somewhat controversial. Bosse and Vestweber (4) originally demonstrated that MAb 10E9.6 can completely block the adhesion of HL-60 promyelocytes to TNF-activated murine endothelial cells in culture and that it reduces thioglycollate-induced neutrophil migration into the peritoneum of BALB/c mice by 63%. Ramos et al. (29), on the other hand, reported that MAb 10E9.6 (and another E-selectin-specific MAb, 9A9) does not block leukocyte rolling in TNF-stimulated venules of BALB/c mice and that MAb 10E9.6 does not affect the adhesion of myeloid cells to E-selectin transfectants. To determine whether MAb 10E9.6 possesses the potential to inhibit neutrophil-endothelial cell interactions in our murine model of I/R, we examined the influence of this MAb on the adhesion of freshly isolated murine (C57BL/6) neutrophils to TNF-activated endothelial cells cultured from C57BL/6 mice. The mouse lung microvascular endothelial cells were stimulated for 4 h with TNF-Radioiodination of monoclonal antibodies. The binding (10E9.6) and nonbinding (P-23) MAbs were labeled with 125I and 131I (NEN, Boston, MA), respectively, using the iodogen method. In brief, iodogen (Sigma T-0656) was dissolved in chloroform at a concentration of 0.5 mg/ml and 250 µl of this solution was placed in glass tubes and evaporated under nitrogen. A 250-µg sample of MAb was added to each iodogen-coated tube, and either 125I or 131I with a total activity of 250 µCi was added. The mixture was incubated on ice, with periodic stirring for 10 min. The total volume was brought to 2.5 ml by adding PBS (pH = 7.4). After radioiodination, the coupled MAb was separated from free 125I or 131I by gel filtration on a Sephadex PD-10 column (Pharmacia Biotech). The column was equilibrated with PBS containing 1% bovine serum albumin and eluted with the same buffer. Two 2.5-ml fractions were collected, the second of which contained the radiolabeled antibody. Absence of free 125I or 131I was ensured by extensive dialysis of the protein-containing fraction. Less than 1% of the activity of the protein fraction was recovered from the dialysis fluid. Labeled MAbs were stored at 4°C.
Animal procedures.
All experimental protocols were applied to either C57BL/6 (wild type)
mice (Jackson Laboratories, Bar Harbor, ME), interferon- [C57Bl/6-Ifg-KO]-deficient mice (Jackson Laboratories), or
transgenic mice with the human gene for Cu,Zn-superoxide dismutase
(SOD) [TgN(SOD1)3Cje]. These Cu,Zn-SOD transgenic mice
(11), with 1.5- to 3.0-fold increases in Cu,Zn-SOD activity in
different tissues, were developed on a C57Bl/6 background and bred by
our animal care facility. Both hemizygous positive (SOD-Tg) and
negative (SOD-nonTg) mice were used in this study. The C57BL/6-Ifg-KO
mice were generated by replacing the one normal Ifg-
gene in mouse embryonic stem cells with a defective allele (6). A total of 111 mice
were used in the study. The experimental procedures described were
reviewed and approved by the Institutional Animal Care and Use
Committee of Louisiana State University Medical Center.
E-selectin expression. A mixture of 10 µg of 125I-labeled E-selectin MAb (10E6) and a dose (0.5-5.0 µg) of 131I-labeled nonbinding MAb (P-23) was injected through the jugular vein catheter. A blood sample was obtained through the carotid artery catheter 5 min after injection of the MAb mixture. The animals were then heparinized (30 units heparin sodium) and rapidly exsanguinated by perfusion of bicarbonate-buffered saline (BBS) through the jugular vein catheter with simultaneous blood withdrawal through the carotid artery catheter. This was followed by perfusion of 10 ml of BBS through the carotid artery catheter after the inferior vena cava was severed at the thoracic level. The small intestine (from ligament of Treitz to ileocecal junction), liver, and lung were harvested and weighed.
The method for calculating E-selectin expression has been described previously (8, 26). In brief, the 125I (binding MAb) and 131I (nonbinding MAb) activities in different tissues and in 50-µl samples of cell-free plasma were counted in a 14800 Wizard 3 gamma-counter (Wallac, Turku, Finland) with automatic correction for background activity and spillover. A 2-µl aliquot of the radiolabeled MAb mixture was assayed to determine total injected activity of each labeled MAb. The radioactivities remaining in the tube used to mix the MAbs and the syringe used to inject the mixture were subtracted from the total injected activity. The accumulated activity of each MAb in an organ was expressed as the percentage of the injected activity per gram of tissue. E-selectin expression was calculated by subtracting the accumulated activity per gram of tissue of the nonbinding MAb (131I-P-23) from the activity of the binding anti-E-selectin MAb (125I-10E9.6). This value, expressed as percent injected dose per gram of tissue, was converted to nanograms of MAb per gram of tissue by multiplying the above value by the total injected binding MAb. Previous studies have shown that MAbs retain their functional activity after radioiodination, as evidenced by a similar effectiveness of labeled and nonlabeled MAbs to block white blood cell adherence in rat mesenteric venules (27). In addition, we have shown (10) that constitutive and endotoxin-induced expression of E-selectin is not detectable in the small intestine and other organs of E-selectin-deficient mice, unlike their wild-type counterparts.Tissue MPO activity.
Samples of small intestine were obtained either in the control period
or after reperfusion, rinsed with cold PBS, blotted dry, and
immediately frozen with liquid nitrogen. The samples were stored at
80°C until being thawed for MPO activity determination using
methods previously described (15). In brief, the tissues were
homogenized in 20 mM phosphate buffer (pH 7.4) and centrifuged at
10,000 rpm for 20 min at 4°C. The pellet was rehomogenized with an
equivalent volume of 50 mM phosphate buffer (pH 6.0) containing 10 mM
EDTA and 0.5% hexadecyltrimethylammonium bromide. MPO (an index of
neutrophil infiltration into tissues) was determined by measuring the
H2O2-dependent oxidation of
3,3',5,5'-tetramethylbenzidine and expressed as units per
gram of wet weight.
Electrophoretic mobility shift assay.
Changes in intestinal DNA binding activity of NF-B activity were
determined using electrophoretic mobility shift assays (EMSAs). Nuclear extracts were prepared from isolated nuclei as previously described (35). Double-stranded DNA nucleotides containing the consensus
B motif (underlined)
and
-
were
labeled with [32P]dATP in the presence of the
Klenow fragment of DNA polymerase 1. The assay was performed in 15 µl
containing 10 µg of nuclear extract protein, binding buffer (10 mM
Tris pH 7.5, 20 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 12% glycerol,
1 µg poly dI-dC, and 1 µg denatured salmon testis DNA), and 1 × 105 counts/min 32P-labeled DNA, as
described by Read et al. (30). The mixture was incubated at room
temperature for 10 min and analyzed by electrophoresis on a 5%
nondenaturing polyacrylamide gel. After electrophoresis, gels were
dried and the protein-DNA complexes were visualized by a PhosphorImager
and quantitated with ImageQuant software (Molecular Dynamics,
Sunnyvale, CA). For supershift analysis, nuclear extracts were
incubated with 1 µl of antiserum against the NF-
B subunit of p50,
p65, or c-Rel at 4°C for 1 h before the addition of binding buffer
and labeled DNA. Antibodies for p50 and c-Rel were obtained from Santa
Cruz Biotech (Santa Cruz, CA), and anti-p65 was purchased from Rockland
Chemicals (Gilbertsville, PA).
Experimental protocols.
To evaluate the magnitude and kinetics of I/R-induced E-selectin
expression, the small intestine was exposed to variable durations of
both ischemia (0-60 min) and reperfusion (3-24 h) as
described above. On the basis of these experiments, optimal periods of
ischemia (45 min) and reperfusion (5 h) were chosen for the
subsequent mechanistic studies. Hence, E-selectin expression was
measured in wild-type, Ifg-KO, SOD-Tg, and SOD-nonTg (genotype-negative littermates of Cu,Zn-SOD transgenic mice) mice exposed to 45 min of
intestinal ischemia followed by 5 h of reperfusion. To define the contributions of TNF- to the I/R-induced expression of
E-selectin, wild-type mice (n = 5) received a blocking antibody
against murine TNF-
(TN3; 20 mg/kg iv immediately before
ischemia) (7). The role of NF-
B was assessed by treatment
with either a proteasome inhibitor (PS341; 0.3 mg/kg iv, 1 h before
ischemia; n = 5) or the antioxidant pyrrolidine
dithiocarbamate (PDTC; 250 mg/kg ip, 1 h before ischemia;
n = 5) (5). In those studies designed to address the relative
contribution of E- and P-selectin to the neutrophil recruitment (MPO)
elicited by intestinal I/R, wild-type mice received an intravenous dose
(2 mg/kg) of a blocking MAb directed against either E-selectin (10E9.6)
or P-selectin (RB40.34) or a combination of the two MAbs. The MAb(s)
was administered 5 min before the induction of ischemia, and
samples for MPO determination were obtained at 5 h after reperfusion.
Statistical evaluation. All values are expressed as means ± SE. Data were compared using an ANOVA with Fisher's protected least significant difference post hoc test. Statistical significance was set at P < 0.05.
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RESULTS |
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Characterization of reperfusion-induced E-selectin expression.
Figure 1A illustrates the time
course of changes in E-selectin expression in mouse small intestine
exposed to 45 min of ischemia and 3-24 h of reperfusion. A
significant elevation in E-selectin expression was noted within 3 h
after reperfusion, with peak expression occurring at 5 h of
reperfusion. Although the value at 8 h was significantly higher than
that measured in sham animals, no difference was noted between the 24 h
and sham values. Figure 1B summarizes the change in E-selectin
expression induced by different durations of ischemia followed
by 5 h of reperfusion, which was previously shown to yield peak
expression of E-selectin. These responses indicate that the magnitude
of E-selectin expression after reperfusion is dependent on the duration
of ischemia. Although 45 min and 60 min of ischemia
produced similar E-selectin responses, the survival rate for the 45-min
occlusion period was ~90%, compared with ~50% for the 60-min
occlusion period. Hence, all subsequent experiments were carried out
using 45-min ischemia and 5-h reperfusion.
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Role of superoxide.
Figure 2 compares the I/R-induced changes
in E-selectin expression in wild-type and SOD-Tg mice. Although I/R
elicited an increased expression of E-selectin in both wild-type and
SOD-Tg mice, E-selectin expression in the SOD-Tg group was 43% lower than the value obtained in wild-type mice. The genotype-negative littermates (SOD-nonTg) of the SOD transgenic mice responded to I/R in
a manner very similar to that of wild-type mice.
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Role of cytokines.
Figure 3 summarizes the effects of I/R on
E-selectin expression in the small intestine of wild-type mice,
wild-type mice treated with a TNF--blocking MAb, and Ifg-KO mice.
Although I/R-induced upregulation of E-selectin did not differ between
wild-type and Ifg-KO mice, treatment of wild-type mice with a
TNF-
-blocking MAb significantly (43%) attenuated I/R-induced
E-selectin expression. These observations suggest that TNF-
, but not
interferon, contributes to the increased E-selectin expression elicited
by intestinal I/R.
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Role of NF-B.
In control intestine, NF-
B activity was very low (Fig.
4). Ischemia (45 min) without
reperfusion did not alter NF-
B activity; however, within 30 min
after reperfusion of the ischemic intestine, NF-
B activation was
noted. NF-
B activity peaked at 60 min and remained elevated above
the control value at 5 h after reperfusion (Fig. 4). The radioactive
signals for the NF-
B-DNA complex was not detected in the presence of
a 100-fold concentration of competitor, indicating specific DNA-protein
binding. Supershift assays showed that anti-p50 antibodies produced a
supershift band that corresponded with the disappearance of the
original protein-DNA complex, indicating that p50 subunits, either as
homodimers or heterodimers with other members of the Rel proteins, were
activated. Both anti-p65 and anti-c-Rel antibodies failed to
produce a supershift band (Fig. 4), suggesting that p65 and c-Rel
subunits are not part of the activation complex.
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Contribution of selectins to I/R-induced granulocyte recruitment.
To assess the contribution of selectins to the recruitment of
granulocytes in postischemic mouse intestine, MPO activity was measured
in intestinal samples obtained at 5 h of reperfusion in wild-type mice
receiving a MAb against E-selectin or P-selectin or a combination of
the two MAbs. In wild-type mice not receiving a MAb, a fivefold
increase in MPO was elicited by I/R (Fig.
6). Although a trend for reduced neutrophil
accumulation was noted in mice treated with the P-selectin MAb, this
did not achieve statistical significance. However, the E-selectin MAb
reduced the I/R-induced MPO value by 60%. Treatment with a combination of the two MAbs tended to lower the MPO response below that observed for E-selectin immunoneutralization alone; however, this did not achieve statistical significance. These findings indicate that E-selectin is a major molecular determinant of the granulocyte recruitment that is observed at 5 h after reperfusion of the ischemic intestine.
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DISCUSSION |
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A major objective of this study was to determine the kinetics and magnitude of E-selectin expression in the intestinal microvasculature after I/R. Although H/R-induced upregulation of E-selectin on HUVECs has been extensively characterized (17, 25), there is relatively little quantitative information concerning how I/R affects the expression of this endothelial CAM in vivo. Several lines of evidence in the literature support the possibility that I/R elicits a significant increase in the expression of E-selectin in different regional vascular beds subjected to an ischemic insult. 1) Plasma levels of circulating soluble E-selectin, which is shed from the surface of activated endothelium, are elevated after myocardial I/R (28). 2) E-selectin mRNA levels are increased in postischemic tissues (33). 3) Immunohistochemical detection of E-selectin reveals an increased expression that is confined to endothelial cells lining postcapillary venules (34, 36). Although these findings suggest that E-selectin biosynthesis and its expression on the surface of endothelial cells are likely increased after I/R, the precision of these approaches does not allow for meaningful quantitative estimates of the changes in E-selectin expression that occur in intact vessels. In the present study, the dual-radiolabeled MAb technique (19, 27) was used to quantify the expression of E-selectin in the postischemic intestinal vasculature. This method has been shown to provide accurate and reproducible estimates of selectin expression in different regional vascular beds (2, 8, 10).
Our estimates of E-selectin expression using the dual-radiolabeled MAb method indicate that I/R elicits an increased expression that is dependent on the duration of both the ischemic insult and the reperfusion period. For a given period of reperfusion (5 h), the magnitude of the E-selectin upregulation was significantly greater after a 45-min ischemic insult compared with a 30-min insult. However, increasing the ischemic duration to 60 min did not yield a larger response, suggesting that the stimulus for upregulation of this endothelial CAM in murine small intestine is maximal at 45 min of ischemia. Our effort to define the kinetics of E-selectin expression after I/R revealed that peak expression is achieved at 5 h, with a return to basal values at 24 h. This time course of E-selectin expression in the postischemic intestine is very similar to the kinetics of E-selectin expression recently described for HUVECs exposed to I/R (17). Furthermore, the time of peak I/R-induced E-selectin expression in mouse intestine is identical to that previously reported (using the dual-radiolabeled MAb method) for P-selectin in postischemic mouse intestine (8). However, in contrast to E-selectin, there is an early (10-30 min), significant increase in P-selectin expression after I/R.
The second major objective of this study was to define the
contributions of superoxide, NF-B, and certain cytokines (TNF-
, interferon-
) to I/R-induced E-selectin expression. Studies on HUVEC
monolayers exposed to H/R suggest that the increased E-selectin expression elicited by I/R is linked to oxidant-mediated activation of
the nuclear transcription factor NF-
B (17). Using transgenic mice
that overexpress Cu,Zn-SOD (11), we have provided evidence that the
enhanced superoxide production that is associated with reperfusion of
the ischemic intestine (22) is an important signal for the increased
biosynthesis and expression of E-selectin in the postischemic
intestinal microvasculature. E-selectin expression in the gut of SOD-Tg
mice was 55% of the value measured in wild-type mice. We previously
reported (8) that the rapid (10-30 min) I/R-induced upregulation
of P-selectin does not differ between SOD-Tg and wild-type mice. Hence,
our findings, coupled with previously published observations, suggest
that superoxide (or a superoxide-derived oxidant) is a more potent
stimulus for transcription-dependent upregulation of E-selectin than
for mobilization of preformed P-selectin to the endothelial cell surface.
A variety of cytokines, including TNF- and interferon-
, are
released from postischemic tissues (16, 32). Many of these cytokines
elicit the translocation of NF-
B into the nucleus, where it binds to
the promoter region of genes for inflammatory molecules such as
E-selectin (30, 31). In the present study, we assessed the contribution
of TNF-
and interferon-
to I/R-induced E-selectin expression in
the intestinal vasculature. Administration of a TNF-
-blocking MAb,
which was previously shown to be effective in attenuating
endotoxin-induced E-selectin expression in murine intestine (9),
reduced I/R-induced E-selectin expression by ~45%, suggesting that
this cytokine is another important stimulus for E-selectin biosynthesis
and expression in postischemic intestine. The comparable E-selectin
responses of wild-type and Ifg-KO mice to I/R suggests that
interferon-
does not contribute significantly to the E-selectin response.
Both oxidants and TNF- are known to promote the biosynthesis of
E-selectin in HUVECs by promoting the nuclear translocation of an
active, binding heterodimeric form (p50/p65) of NF-
B (30, 31).
HUVECs exposed to H/R exhibit nuclear translocation of p50/p65 with a
corresponding increase in E-selectin expression (17). Furthermore,
experimental strategies directed toward interfering with the activation
and/or upregulation of p50/p65, such as treatment with antioxidants
(PDTC), proteasome inhibitors (PS341) or ds-oligonucleotides containing
the NF-
B cognate sequence, effectively attenuate the increased
E-selectin expression observed on HUVECs exposed to H/R (17) or TNF-
(18). In the present study, we have demonstrated significant nuclear
translocation of NF-
B within 1 h after reperfusion of ischemic
murine intestine. However, unlike posthypoxic HUVECs, which exhibit
nuclear translocation of p50/p65, the dominant dimeric form of NF-
B
that was detected in postischemic intestine is p50/p50. The
contribution of the p50/p50 homodimer to activation of the E-selectin
gene remains unclear; the available evidence favors a role for p50/p65
in this response (30, 31). It should be noted, however, that our
inability to detect p50/p65 in whole homogenates of postischemic
intestine does not exclude the possibility that this heterodimer is
activated and upregulated in vascular endothelial cells, which comprise
>5% of total intestinal mass.
To further address the possible involvement NF-B in I/R-induced
E-selectin expression, we treated some mice with agents that have
previously been shown to interfere with NF-
B-mediated E-selectin expression on posthypoxic HUVECs. Ichikawa et al. (17) reported that
HUVEC monolayers pretreated with either a proteasome inhibitor (MG132)
or an antioxidant (PDTC) significantly attenuated H/R-induced E-selectin expression. In the present study, we observed that the
proteasome inhibitor PS341 reduced I/R-induced E-selectin expression in
mouse intestine by ~36%, which compares with the 58% reduction in
E-selectin expression observed in posthypoxic HUVECs (17). These
findings with the proteasome inhibitor suggest that NF-
B may well
provide a linkage between superoxide and TNF-
and the increased
E-selectin expression induced by I/R. However, in contrast to the
previously published in vitro data, the dithiocarbamate PDTC was
ineffective in blunting the upregulation of E-selectin observed in our
in vivo model of I/R. A definitive explanation for the ineffectiveness
of PDTC in vivo is not readily available, but it may reflect a limited
bioavailability of the compound to vascular endothelial cells in vivo
or the fact that PDTC can act as a prooxidant because of its ability to
chelate and transport certain transition metals (e.g., copper) into
cells (14). Furthermore, PDTC has been shown to inactivate
intracellular antioxidant enzymes such as SOD and glutathione
peroxidase, thereby facilitating the intracellular accumulation of
reactive oxygen species (14).
Another major objective of this study was to assess the importance of E-selectin in mediating I/R-induced neutrophil recruitment. Although there are several published reports that implicate P-selectin as a mediator of I/R-induced leukocyte recruitment (13, 26), there are relatively few data that support a role for E-selectin in the same recruitment process (1). The latter situation likely results from two factors: 1) a focus of most I/R studies on the inflammatory events that occur within the first hour after reperfusion, and 2) the use of anti-human MAbs with limited binding affinity to E-selectin expressed in rodent microvessels (21). In the present study, we determined whether an anti-murine E-selectin MAb that blocks the adhesion of murine neutrophils to monolayers of TNF-activated murine microvascular endothelial cells alters the accumulation of neutrophils (MPO) observed in murine small intestine at 5 h after reperfusion, i.e., at the time of peak E-selectin expression. We found that immunoneutralization of E-selectin reduced I/R-induced neutrophil accumulation by 60%, whereas administration of a blocking MAb to murine P-selectin did not significantly alter this response. These observations indicate that the time-dependent increase in E-selectin expression observed in postischemic intestine is a functionally significant response that accounts for much of the I/R-induced neutrophil accumulation at 5 h after reperfusion. However, our findings do not necessarily implicate E-selectin as the principal rolling receptor in postischemic intestinal microvasculature because there is published evidence that suggests that E-selectin serves a function, other than rolling, that appears to be important for neutrophil recruitment to inflammatory sites in mice (29).
In conclusion, this study demonstrates that intestinal I/R is
accompanied by an increased E-selectin expression that is dependent on
both the duration of the ischemic insult and period of reperfusion. Superoxide and TNF- contribute to the I/R-induced E-selectin upregulation, in part because of the activation of NF-
B. The increased E-selectin expression accounts for most of the neutrophil accumulation that occurs several hours after reperfusion.
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ACKNOWLEDGEMENTS |
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D. N. Granger (P01-DK-43785 and R01-HL-26441), M. B. Grisham (P01-DK-43785 and R01-DK-47663), J. S. Alexander (P01-DK-43785), and C. J. Epstein (P01-AG-08938 and R01-AG-16998) are supported by grants from the National Institutes of Health.
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. N. Granger, Dept. of Molecular and Cellular Physiology, LSU Medical Ctr., 1501 Kings Hwy., Shreveport, LA 71130-3932 (E-mail: dgrang{at}lsumc.edu).
Received 13 September 1999; accepted in final form 26 January 2000.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Altavilla, D,
Squadrito F,
Ioculano M,
Canale P,
Campo GM,
Zingerelli B,
and
Caputi AP.
E-selectin in the pathogenesis of experimental myocardial ischemia-reperfusion injury.
Eur J Pharmacol
270:
45-51,
1994[Medline].
2.
Bauer, P,
Russell JM,
and
Granger DN.
Role of endotoxin in intestinal reperfusion-induced expression of E-selectin.
Am J Physiol Gastrointest Liver Physiol
276:
G479-G484,
1999
3.
Binns, RM,
Licence ST,
Harrison AA,
Keelan ET,
Robinson MK,
and
Haskard DO.
In vivo E-selectin upregulation correlates with infiltration of PMN, later with PBL entry: MABs block both.
Am J Physiol Heart Circ Physiol
270:
H183-H193,
1996
4.
Bosse, R,
and
Vestweber D.
Only simultaneous blocking of the L- and P-selectin completely inhibits neutrophil migration into mouse peritoneum.
Eur J Immunol
24:
3019-3024,
1994[ISI][Medline].
5.
Conner, EM,
Brand S,
Davis JM,
Laroux FS,
Palombella VJ,
Fuseler JW,
Kang DY,
Wolf RE,
and
Grisham MB.
Proteasome inhibitor attenuates nitric oxide synthase expression, VCAM-1 transcription, and the development of chronic colitis.
J Pharmacol Exp Ther
282:
1615-1622,
1997
6.
Doherty, GM,
Lange JR,
Langstein HN,
Alexander HR,
Buresh CM,
and
Norton JA.
Evidence for IFN- as a mediator of the lethality of endotoxin and tumor necrosis factor-
.
J Immunol
149:
1666-1670,
1992
7.
Eppihimer, MJ,
and
Granger DN.
Ischemia/reperfusion-induced leukocyte-endothelial interactions in postcapillary venules.
Shock
8:
16-25,
1997[ISI][Medline].
8.
Eppihimer, MJ,
Russell J,
Anderson DC,
Epstein CJ,
Laroux S,
and
Granger DN.
Modulation of P-selectin expression in the postischemic intestinal microvasculature.
Am J Physiol Gastrointest Liver Physiol
273:
G1326-G1332,
1997
9.
Eppihimer, MJ,
Russell J,
Langley R,
Gerritsen M,
and
Granger DN.
Role of tumor necrosis factor and interferon gamma in endotoxin-induced E-selectin expression.
Shock
11:
93-97,
1999[ISI][Medline].
10.
Eppihimer, MJ,
Wolitzky BA,
Anderson DC,
Labow MA,
and
Granger DN.
Heterogeneity of E-and P-selectin expression in vivo.
Circ Res
79:
560-569,
1996
11.
Epstein, CJ,
Avraham KB,
Loverr M,
Smith S,
Elroy-stein O,
Rotman G,
Bry C,
and
Groner Y.
Transgenic mice with increased CuZn-superoxide dismutase activity: animal model of dosage effects in Down syndrome.
Proc Natl Acad Sci USA
84:
8044-8048,
1987[Abstract].
12.
Granger, DN.
Cell Adhesion and Migration. II. Leukocyte-endothelial cell adhesion in the digestive system.
Am J Physiol Gastrointest Liver Physiol
273:
G982-G986,
1997
13.
Granger, DN,
and
Korthuis RJ.
Physiologic mechanisms of postischemic tissue injury.
Annu Rev Physiol
57:
311-332,
1995[ISI][Medline].
14.
Grisham, MB.
NF-B activation in acute pancreatitis: protective, detrimental, or inconsequential?
Gastroenterology
116:
489-492,
1999[ISI][Medline].
15.
Grisham, MB,
Benoit JN,
and
Granger DN.
Assessment of leukocyte involvement during ischemia and reperfusion of the intestine.
Methods Enzymol
186:
729-742,
1990[Medline].
16.
Grotz, MRW,
Ding J,
Guo W,
Huang Q,
and
Deitch EA.
Comparison of plasma cytokine levels in rats subjected to superior mesenteric artery occlusion or hemorrhagic shock.
Shock
3:
362-368,
1995[ISI][Medline].
17.
Ichikawa, H,
Flores S,
Kvietys PR,
Wolf RE,
Yoshikawa T,
Granger DN,
and
Aw TY.
Molecular mechanisms of anoxia/reoxygenation-induced neutrophil adherence to cultured endothelial cells.
Circ Res
81:
922-931,
1997
18.
Kalogeris, TJ,
Laroux FS,
Cockrell A,
Ichikawa H,
Okayama N,
Phifer TJ,
Alexander JS,
and
Grisham MB.
Effect of selective proteasome inhibitors on TNF-induced activation of primary and transformed endothelial cells.
Am J Physiol Cell Physiol
276:
C856-C864,
1999
19.
Keelan, ET,
Licence ST,
Peters AM,
Binns RM,
and
Haskard DO.
Characterization of E-selectin expression in vivo with use of a radiolabeled monoclonal antibody.
Am J Physiol Heart Circ Physiol
266:
H278-H290,
1994[Abstract].
20.
Kokura, S,
Wolf RE,
Yoshikawa T,
Granger DN,
and
Aw TY.
Molecular mechanisms of neutrophil-endothelial cell adhesion induced by redox imbalance.
Circ Res
84:
516-524,
1999
21.
Kurose, I,
Anderson DC,
Miyasaka M,
Tamatani T,
Paulson JC,
Todd RF,
Rusche JR,
and
Granger DN.
Molecular determinants of reperfusion-induced leukocyte adhesion and vascular protein leakage.
Circ Res
74:
336-343,
1994[Abstract].
22.
Kurose, I,
Wolf RE,
Grisham MB,
and
Granger DN.
Hypercholesterolemia enhances oxidant production in mesenteric venules exposed to ischemia-reperfusion.
Arterioscler Thromb Vasc Biol
18:
1583-1588,
1998
23.
Kvietys, PR,
and
Granger DN.
Endothelial cell monolayers as a tool for studying microvascular pathophysiology.
Am J Physiol Gastrointest Liver Physiol
273:
G1189-G1199,
1997
24.
Ma, L,
Raycroft L,
Asa D,
Anderson DC,
and
Geng JB.
A sialoglycoprotein from human leukocyte function as a ligand for P-selectin.
J Biol Chem
269:
27739-27746,
1994
25.
Palluy, O,
Morliere L,
Gris JC,
Bonne C,
and
Modat G.
Hypoxia/reoxygenation stimulates endothelium to promote neutrophil adhesion.
Free Radic Biol Med
13:
21-30,
1992[ISI][Medline].
26.
Panes, J,
and
Granger DN.
Leukocyte-endothelial cell interactions: molecular mechanisms and implications in gastrointestinal disease.
Gastroenterology
114:
1066-1090,
1998[ISI][Medline].
27.
Panes, J,
Perry MA,
Anderson DC,
Manning A,
Leone B,
Cepinskas G,
Rosenbloom CL,
Miyasaka M,
Kvietys PR,
and
Granger DN.
Regional differences in constitutive and induced ICAM-1 expression in vivo.
Am J Physiol Heart Circ Physiol
269:
H1955-H1964,
1995
28.
Pudil, R,
Pidrman V,
Krejsek V,
Gregor J,
Tichy M,
Andrys C,
and
Drahosova M.
Cytokines and adhesion molecules in the course of acute myocardial infarction.
Clin Chim Acta
280:
27-134,
1999.
29.
Ramos, CL,
Kunkel EJ,
Lawrence MB,
Jung U,
Vestweber D,
Bosse R,
McIntyre KW,
Gillooly KM,
Norton CR,
Wolitzky BA,
and
Ley K.
Differential effect of E-selectin antibodies on neutrophil rolling and recruitment to inflammatory sites.
Blood
15:
3009-3018,
1997.
30.
Read, MA,
Neish AS,
Luscinskas FW,
Palombella VJ,
Maniatis T,
and
Collins T.
The proteasome pathway is required for cytokine-induced endothelial-leukocyte adhesion molecule expression.
Immunity
2:
493-506,
1995[ISI][Medline].
31.
Read, MA,
Whitley MZ,
Williams AJ,
and
Collins T.
NF-B and I
B: an inducible regulatory system in endothelial activation.
J Exp Med
179:
503-512,
1994[Abstract].
32.
Schwartz, MD,
Repine JE,
and
Abraham E.
Xanthine oxidase-derived radicals increase lung cytokine expression in mice subjected to hemorrhagic shock.
Am J Respir Cell Mol Biol
12:
434-440,
1995[Abstract].
33.
Stotland, MA,
and
Kerrigan CL.
E- and L-selectin adhesion molecules in musculocutaneous flap reperfusion injury.
Plast Reconstr Surg
99:
2010-2020,
1997[ISI][Medline].
34.
Wang, C,
Kerrigan CL,
and
Stotland MA.
Kinetics of E-selectin expression in surgical flaps.
Plast Reconstr Surg
100:
1482-1488,
1997[ISI][Medline].
35.
Yeh, KY,
Yeh M,
and
Glass J.
Expression of rat intestinal brush-border membrane hydrolases and ferritin following segmental ischemia-reperfusion.
Am J Physiol Gastrointest Liver Physiol
275:
G572-G583,
1998
36.
Zhang, R,
Chopp M,
Zhang Z,
Jiang N,
and
Powers C.
The expression of P- and E-selectin in three models of middle cerebral artery occlusion.
Brain Res
785:
207-214,
1998[ISI][Medline].