XO increases neutrophil adherence to endothelial
cells by a dual ICAM-1 and P-selectin-mediated mechanism
LANCE S. TERADA, BROOKS M. HYBERTSON, KEVIN G. CONNELLY,
DAVID WEILL, DALE PIERMATTEI, AND JOHN E. REPINE
Webb-Waring Institute for Biomedical Research and Department of Medicine,
University of Colorado Health Sciences Center, Denver, Colorado 80262
xanthine oxidase; platelet activating factor; CD18; CD11b;
Mac-1; CD11a; leukocyte functional antigen-1; heparin; hydrogen peroxide; multiorgan failure; acute respiratory distress
syndrome
MULTIORGAN FAILURE IS A SYNDROME characterized by the
dissemination of inflammation from one organ to another, prompting its more recent moniker, systemic
inflammatory response syndrome. The hallmark of this
disorder is the attachment of neutrophils to endothelium in a number of organs. Indeed, a localized primary
injury causes neutrophil recruitment and inflammation in a secondary organ in a number of experimental
models. For instance, reperfusion injury to hindlimbs
(14) or intestines (33) produces an inflammatory lung
injury, and focal injury to one lung causes inflammatory
changes in the opposite lung (3).
Although the mechanism by which the secondarily
affected organ initially retains neutrophils is not clearly
understood, the prooxidant enzyme xanthine oxidase
(XO) may be pivotal. Endogenous XO participates in
postischemic neutrophil adherence in vivo (10) and
866
reoxygenation-induced neutrophil adherence in vitro
(34). In addition, circulating levels of XO are elevated in
patients with acute lung injury (11) and after limb
ischemia (8), and infused XO increases neutrophil
adherence to rat mesenteric venules (9). We have
recently shown that circulating XO increases and mediates neutrophil retention in lungs after intestinal ischemia (33), highlighting the potential role of XO in
initiating distant organ inflammation. Accordingly, the
goal of this study was to identify pathways involved in
extracellular XO-mediated neutrophil adherence to endothelial cells (EC).
MATERIALS AND METHODS
Source of reagents. Platelet-activating factor (PAF; from
bovine heart lecithin), superoxide dismutase (SOD; bovine
erythrocyte, 3,000 U/mg) and XO (grade III from bovine milk,
1.2 U/mg) were obtained from Sigma Chemical (St. Louis,
MO). Blocking monoclonal antibodies against CD11a and
intercellular adhesion molecule-1 (ICAM-1; CD54) were from
AMAC (Westbrook, ME). Blocking monoclonals against CD11b
were from Sigma Chemical, and blocking monoclonals against
P-selectin (CD62) were from Monosan. Each antibody was of
subtype immunoglobulin G1 (IgG1 ), and, therefore, a mouse
IgG1 isotype control (derived from the MOPC-21 tumor line)
was obtained from Sigma Chemical. Unconjugated and peroxidase-conjugated rabbit polyclonal antibodies to human von
Willibrand factor (vWF) was from Dako (Carpinteria, CA).
WEB-2086 was a kind gift of Boehringer Ingelheim (Ridgefield, CT). Heparin (porcine intestinal mucosa) was purchased from SoloPak Laboratories (Elk Grove Village, IL).
4-Hydroxy-2,2,6,6-tetramethylpiperidinyloxy (Tempol) was obtained from Aldrich (Milwaukee, WI). Rat interferon (IFN)
-a/b (3.5 3 106 IU/mg) was obtained from Lee Bio Molecular
(San Diego, CA). Catalase (bovine liver, 81,536 U/mg) was
purchased from Worthington Biochemical (Freehold, NJ).
[3H]acetate (.500 mCi/mmol) was from New England Nuclear
(Boston, MA). Hypoxanthine (HX) and all other reagents
were obtained from Sigma Chemical.
Endothelial cell culture. Bovine pulmonary artery EC were
harvested by using collagenase digestion (34). After two
passages in D-valine (Sigma Chemical) to minimize smooth
muscle contamination, EC were cultured in Eagle’s minimum
essential medium with 10% fetal calf serum and studied after
three to five passages. Cultures were found to be .99.5% EC
by vWF and low-density lipoprotein receptor staining.
Static neutrophil adherence. Neutrophils were isolated
from healthy human donors and labeled with 51Cr, as previously described (34). EC were plated in 24-well plates and
uniformly used at 1–2 days after reaching confluence. EC
were washed twice with Hanks’ balanced salt solution (HBSS)
and incubated with various combinations of XO (10 mU/ml),
HX (200 µM), heparin (50 U/ml), catalase (815 U/ml), SOD (30
U/ml), Tempol (5 mM), WEB-2086 (1 mM), reagent PAF (1 µM),
or monoclonal antibodies (4 µg/ml), in HBSS, for 30 min at
0161-7567/97 $5.00 Copyright r 1997 the American Physiological Society
Downloaded from http://jap.physiology.org/ by 10.220.33.1 on June 17, 2017
Terada, Lance S., Brooks M. Hybertson, Kevin G.
Connelly, David Weill, Dale Piermattei, and John E.
Repine. XO increases neutrophil adherence to endothelial
cells by a dual ICAM-1 and P-selectin-mediated mechanism.
J. Appl. Physiol. 82(3): 866–873, 1997.—Circulating xanthine
oxidase (XO) can modify adhesive interactions between neutrophils and the vascular endothelium, although the mechanisms underlying this effect are not clear. We found that
treatment with XO of bovine pulmonary artery endothelial
cells (EC), but not neutrophils or plasma, increased adherence, suggesting that XO had its primary effect on EC. The
mechanism by which XO increased neutrophil adherence to
EC involved binding of XO to EC and production of H2O2. XO
also increased platelet-activating factor production by EC by
a H2O2-dependent mechanism. Similarly, the platelet-activating factor-receptor antagonist WEB-2086 completely blocked
XO-mediated neutrophil EC adherence. In addition, neutrophil adherence was dependent on the b2-integrin Mac-1
(CD11b/CD18) but not on leukocyte functional antigen-1
(CD11a/CD18). Treatment of EC with XO for 30 min did not
alter intercellular adhesion molecule-1 surface expression
but increased expression of P-selectin and release of von
Willibrand factor. Antibodies against P-selectin (CD62) did
not affect XO-mediated neutrophil adherence under static
conditions but decreased both rolling and firm adhesive
interactions under conditions of shear. We conclude that
extracellular XO associates with the endothelium and promotes neutrophil-endothelial cell interactions through dual
intercellular adhesion molecule-1 and P-selectin ligation, by a
mechanism that involves platelet-activating factor and H2O2
as intermediates.
XANTHINE OXIDASE AND NEUTROPHIL ADHERENCE
Fig. 1. Neutrophil adherence increased after treatment of endothelial cells (EC) with xanthine oxidase (XO; 5–30 mU/ml) and hypoxanthine (HX; 200 µM) for 30 min (* P , 0.01), compared with untreated
EC. Values are means 6 SE of 6 individual determinations.
through the tube with a syringe pump at a constant rate
(0.173 ml/min). A second syringe pump infused HBSS 1 5%
fetal calf serum at a variable rate. From the measured total
flow rate and the cylindrical geometry of the tube, the shear
at the surface of the tube was calculated by using the HagenPoiseuille equation (22). The interaction of neutrophils with EC
was recorded on videocassette recorder (Javelin recorder) for later
playback analysis. The number of rolling neutrophils crossing a
standardized 250-µm bar, neutrophil rolling velocity (average of
6–12 neutrophils), and number of firmly adherent neutrophils
(no movement for at least 30 s) per 0.0625 mm2 were
determined for three random fields per tube.
RESULTS
Effect of XO on neutrophil adherence. Exposure of EC
and neutrophils to XO (5–30 mU/ml) and substrate
(HX) for 10–30 min increased (P , 0.01) neutrophil
adherence to EC monolayers (Figs. 1 and 2). Addition of
substrate alone did not affect (P . 0.05) neutrophil
adherence. Pretreatment of EC, but not neutrophils,
with XO and HX increased (P , 0.001) neutrophil
adherence to EC monolayers (Fig. 3). Addition of plasma
pretreated with XO and HX had no effect on neutrophil
Fig. 2. Adherence of neutrophils to EC monolayers increased after
treatment of EC with XO (10 mU/ml) and HX (200 µM) for 10, 20, or
30 min (* P , 0.001), compared with untreated EC. Treatment of EC
with XO/HX for 40–60 min did not (P . 0.05) affect neutrophil
adherence. Values are means 6 SE of 6 individual determinations.
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37°C. In some experiments, EC were incubated with various
agents in 50% pooled normal human plasma. EC were then
washed twice with HBSS, and unstimulated polymorphonuclear leukocytes (PMN; 106/well) were gently added. In
some experiments, WEB-2086 was added to EC and PMN.
After a 30-min incubation at 37°C, nonadherent neutrophils
were collected and pooled with the first wash. Residual 51Cr in
adherent neutrophils was released with NaOH and quantified by liquid scintigraphy. Adherence was defined as the
percentage of counts per minute (cpm) in the media and first
wash, relative to total cpm in media per wash plus EC (cpm
media 1 first wash/cpm media 1 first wash 1 EC releasate).
Visual inspection under all conditions revealed persistence of
EC monolayers before NaOH lysis. In some experiments,
monolayers were first pretreated with XO and/or heparin for
60 min at 37°C then triply washed and incubated with
neutrophils in the presence of HX. In some experiments,
neutrophils were fixed with paraformaldehyde (1%) for 10
min at 25°C after 51Cr loading.
PAF assay. EC monolayers in 25-cm2 flasks were triply
washed to remove serum, then incubated with 25 µCi 3Hacetate in 1 ml HBSS with 10 mM N-2-hydroxyethylpiperazine-N8-2-ethanesulfonic acid (21) in the presence or absence
of XO (10 mU/ml), HX (200 µM), SOD (30 U/ml), or catalase
(815 U/ml) for up to 30 min. Without aspirating the media,
the reaction was stopped with 0.5 ml of 50 mM acetic acid in
MeOH, and EC were scraped off, pooled with a subsequent
2-ml acetic acid/MeOH wash, and added to 1.25 ml CHCl3.
Labeled PAF was extracted (4) with 10 µg cold PAF as carrier
by using 1.25 ml CHCl3 and 1.25 ml of 0.1 M sodium acetate.
The lower phase was dried under N2 and resuspended in 100
µl CHCl3/MeOH (9:1). An aliquot was removed for assessment
of total [3H]acetate incorporation, and the remainder was
separated by thin-layer chromatography (silica gel 60, Merck,
Darmstadt, Germany) by using CHCl3/MeOH/glacial acetic
acid/H2O (50:25:8:4). Lipids were visualized with iodine vapor, and lanes were scraped off in fractions by using the
mobility of authentic PAF as a guide. After quantification of
3H in these fractions with liquid scintigraphy, the amount of
[3H]acetate incorporated into PAF was calculated (36).
vWF release and P-selectin and ICAM-1 surface expression.
EC were grown to confluence in 96-well enzyme-linked immunosorbent assay (ELISA) plates and exposed to XO (10
mU/ml) and HX (200 µM) in HBSS for 30 min. Control EC
were exposed to IFN-a/b (24,000 U/ml) for 24 h to increase
ICAM-1 expression. EC were then washed twice with HBSS
and blocked with 2% bovine serum albumin (BSA) in HBSS at
25°C for 30 min. After two subsequent washes, EC were
incubated with either anti-P-selectin or anti-ICAM-1 (1:500
in 0.1% BSA) for 30 min at 37°C, washed twice, then
incubated with rabbit anti-mouse IgG-peroxidase (1:1,000 in
0.1% BSA) for 20 min at 25°C. After two subsequent washes,
the plates were developed with o-phenylenediamine dihydrochloride (OPD). Baseline values (EC treated with OPD in the
absence of antibodies) were subtracted from sample values to
account for the low levels of endogenous EC peroxidases. vWF
released into the media was assessed by ELISA as previously
described (13).
Dynamic neutrophil adherence. EC were passaged into
gelatin-coated glass capillary tubes (1.1-mm ID, Scientific
Manufacturing Industries, Emeryville, CA). After attachment of EC, medium was changed once, and EC were allowed
to grow overnight to form confluent monolayers on approximately one-half of the internal surface of the tube. After
treatment of EC with various agents, the capillary tubes were
secured on the stage of an inverted microscope with EC in the
dependent position. Neutrophils (106/ml) were perfused
867
868
XANTHINE OXIDASE AND NEUTROPHIL ADHERENCE
Fig. 3. Neutrophil adherence increased (* P , 0.001)
after treatment of EC with XO (10 mU/ml) and HX (200
µM), compared with untreated EC. Treatment of neutrophils with XO and HX did not alter (P . 0.05) adherence
of neutrophils to EC monolayers. Values are means 6
SE of 6 individual determinations.
Fig. 4. Pretreatment of EC with XO (10 mU/ml),
followed by washing and subsequent addition of HX
(200 µM) increased (* P , 0.001) adherence of neutrophils, compared with EC treated with HX alone. Cotreatment of EC with heparin (Hep; 50 U/ml) and XO
decreased (** P , 0.001) neutrophil adherence, compared with EC pretreated with XO alone. Heparin did
not decrease neutrophil adherence (P . 0.05) when
added to EC simultaneously with both XO and HX.
Values are means 6 SE of 6 individual determinations.
HBSS, Hanks’ balanced salt solution.
4%, P . 0.05). To investigate the importance of XO
binding to EC in a more relevant antioxidant-replete
milieu, we exposed EC to HX and XO in 50% human
plasma. HX and XO increased neutrophil adherence
(P , 0.001) in 50% plasma (Fig. 5). In contrast to the
experiment performed in HBSS alone, heparin decreased neutrophil adherence when added concurrently with HX and XO in 50% plasma (P , 0.001).
Again, heparin had no effect (P . 0.05) on baseline
adherence.
Effect of O2 metabolite scavengers on neutrophil adherence. Catalase decreased (P , 0.001) XO-mediated
adherence to EC monolayers (Fig. 6), whereas SOD or
the membrane-permeable SOD mimic Tempol (25, 32)
did not affect (P . 0.05) XO-mediated neutrophil
adherence. None of these agents altered baseline adherence to non-XO-treated EC (P . 0.05). Conversely,
treatment of EC with H2O2 (10–100 µM) increased (P ,
0.01) neutrophil adherence (Fig. 7).
Effect of XO on PAF production. Treatment of EC
with XO and HX for 30 min increased (P , 0.001)
[3H]acetate incorporation into PAF (Fig. 8). Catalase
(P , 0.01), but not SOD (P . 0.05), decreased [3H]acetate
incorporation in XO-treated EC. [3H]acetate incorpora-
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adherence (not shown). In another experiment, conditioned media from HX/XO-treated EC were added to
untreated EC monolayers. Adherence of neutrophils
was higher (P , 0.01) to HX/XO-treated EC than to EC
exposed to HX/XO-EC conditioned media (control 3.09 6
0.25%; HX/XO 8.13 6 0.25%; conditioned media 4.27 6
0.25%).
Effect of heparin on neutrophil adherence. The ability
of EC-bound XO to modify neutrophil adherence was
assessed by first preincubating monolayers with XO in
the absence of substrate. After removal of unassociated
XO by extensive washing, HX was then added and
resulted in increased (P , 0.001) neutrophil adherence
compared with EC not preincubated with XO (Fig. 4).
Heparin, which interferes with binding of XO to cell
surfaces (1, 31), decreased (P , 0.001) neutrophil
adherence when added with XO to EC in the preincubation phase (Fig. 4). In contrast, heparin did not affect
(P . 0.05) neutrophil adherence when added concurrently with both XO and HX in HBSS. Heparin alone
had no effect (P . 0.05) on neutrophil adherence,
compared with baseline. In addition, heparin had no
effect on phorbol myristate acetate-mediated neutrophil adherence (PMA, 46 6 3%; PMA 1 heparin, 41 6
XANTHINE OXIDASE AND NEUTROPHIL ADHERENCE
869
Fig. 5. EC were exposed to HX and XO in 50%
human plasma. XO (10 mU/ml) and HX (200 µM)
increased neutrophil adherence (* P , 0.001) in 50%
plasma. Heparin decreased neutrophil adherence
when added concurrent with HX and XO in 50%
plasma (** P , 0.001). Heparin had no effect (P .
0.05) on baseline adherence. Values are means 6 SE
of 6 individual determinations.
Fig. 6. Treatment of EC with XO (10 mU/ml) and HX (200 µM)
increased (* P , 0.001) neutrophil adherence compared with untreated EC, and catalase (815 U/ml) decreased (** P , 0.001)
neutrophil adherence to EC exposed to XO and HX. Catalase (Cat)
did not affect neutrophil adherence to untreated EC (P . 0.05).
Superoxide dismutase (SOD; 30 U/ml) and Tempol (5 mM) had no
effect on neutrophil adherence to XO-treated or untreated EC (P .
0.05). Values are means 6 SE of 6 individual determinations.
cyte function-associated antigen-1 (LFA-1), did not
affect XO-mediated adherence (P . 0.05). Irrelevant
isotype antibodies also had no effect on neutrophil
adherence to EC (P . 0.05). Furthermore, fixation of
neutrophil proteins with paraformaldehyde also decreased (P , 0.001) adherence to XO-treated EC (Fig.
11). Surface expression of ICAM-1 did not change after
treatment of EC with XO (control 0.083 6 0.007
absorbance units; XO1HX 0.080 6 0.011, P . 0.05 vs.
control; IFN-a/b 0.222 6 0.033, P , 0.001 vs. control).
In addition, abrogation of de novo protein synthesis
with cycloheximide did not decrease neutrophil adherence to XO-treated EC (XO1HX 10.1 6 0.4%;
XO1HX1cycloheximide 12.3 6 0.6%, P . 0.05). Surface expression of P-selectin increased after treatment
with XO (control 0.216 6 0.040 absorbance units;
XO1HX 0.445 6 0.039, P , 0.01), and release of vWF
also increased after treatment with XO (control 0.030 6
0.021 absorbance units; XO1HX 0.108 6 0.023, P ,
0.05).
Effect of XO on neutrophil rolling behavior. Under
static conditions, antibodies against P-selectin did not
(P . 0.05) affect XO-mediated neutrophil adherence
(Fig. 12). Under shear-loaded conditions, more (P ,
0.001) neutrophils rolled at all three shear rates after
treatment of EC with XO (Fig. 13). Antibodies to
Fig. 7. Treatment of EC with H2O2 (10–100 µM) increased (* P ,
0.01) neutrophil adherence, compared with untreated EC. Values are
means 6 SE of 6 individual determinations.
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tion into PAF also increased after 10 min (4,459 6 294
cpm) and 20 min (3,873 6 636 cpm) of exposure to HX
and XO, compared with control (1,489 6 224 cpm) (P ,
0.01). In parallel, the PAF-receptor antagonist WEB2086 was found to decrease (P , 0.001) XO-mediated
adherence of neutrophils to EC when present during
both XO treatment and incubation with neutrophils
(Fig. 9). In separate experiments, we found that treatment of EC with WEB-2086 only during exposure to XO
and not during subsequent incubation with neutrophils
decreased XO-mediated adherence by only 10 6 9%.
Conversely, treatment of EC with Web 2086 only during
incubation with neutrophils diminished XO-mediated
adherence by 97 6 8%. Addition of PAF also increased
neutrophil adherence to EC, whether added to neutrophils in the presence of EC (Fig. 9) or to EC alone,
before addition of neutrophils (not shown). Web 2086
decreased (P , 0.001) PAF-mediated neutrophil adherence.
Effect of monoclonal antibodies on neutrophil adherence. Antibodies directed against ICAM-1 or CD11b, the
a-subunit of the neutrophil b2-integrin Mac-1, decreased (P , 0.001) neutrophil adherence to XOtreated EC (Fig. 10). In contrast, antibodies against
CD11a, the a-subunit of the neutrophil integrin leuko-
870
XANTHINE OXIDASE AND NEUTROPHIL ADHERENCE
P-selectin decreased rolling behavior (P , 0.05), and
cotreatment with antibodies to P-selectin and ICAM-1
completely blocked XO-mediated neutrophil rolling
(P , 0.001). Antibodies to ICAM-1 alone decreased the
number of rolling neutrophils at the lowest shear rate
(P , 0.05). Treatment of EC with XO also decreased
neutrophil rolling velocity at a shear rate of 38 s21 (P ,
0.01) but not at the higher shear rates. Antibodies to
P-selectin and/or ICAM-1 did not significantly alter
rolling velocity. Finally, XO increased the number of
firmly adherent neutrophils to EC at all three shear
rates (P , 0.01). Antibodies to P-selectin decreased firm
adherence at shear rates of 60 and 96 s21 (P , 0.01) but
not at 38 s21 (P . 0.05). Treatment of EC with antibodies to ICAM-1 alone (P , 0.05) or cotreatment of EC
with antibodies to both P-selectin and ICAM-1 (P ,
0.001) completely suppressed XO-mediated firm adherence at all three shear rates.
DISCUSSION
We recently found that after mesenteric ischemia
plasma levels of XO, an efficient enzymatic source of
Fig. 9. Neutrophil adherence increased (* P , 0.001) after treatment
of EC with either XO (10 mU/ml) plus HX (200 µM) or PAF (1 µM),
compared with untreated EC. Treatment with WEB-2086 (1 mM)
decreased (** P , 0.001) neutrophil adherence to both XO/HX- and
PAF-treated EC. Values are means 6 SE of 6 individual determinations.
reactive oxygen intermediates, increased and mediated
retention of neutrophils in the lung parenchyma (33).
The ability of circulating XO to increase neutrophil-EC
interactions provides an interesting mechanism by
which a primarily injured organ may initiate inflammation in other vascular beds. However, the literature
provides conflicting paradigms of the mechanism by
which prooxidants may promote retention of neutrophils in inflammed tissues. For instance, high levels (10
mM) of H2O2 were found to rapidly incite PAF production by EC (21), whereas PAF did not appear to be
responsible for neutrophil adherence when lower, more
physiological doses of H2O2 were employed (27). In
addition, conclusions diverge regarding whether
H2O2 mediates neutrophil adherence exclusively by
P-selectin- (27) or ICAM-1-related mechanisms (23).
Similarly, conflicting data exist regarding the mechanism by which XO alters neutrophil adherence (9, 29).
We found evidence to suggest that extracellular XO
promotes neutrophil adherence to EC by a mechanism
involving the production of PAF by EC and the binding
of both ICAM-1 and P-selectin to neutrophil ligands.
We found first that XO increased neutrophil adherence
Fig. 11. Neutrophil adherence increased (* P , 0.001) after treatment of EC with XO (10 mU/ml) and HX (200 µM). Pretreatment of
neutrophils with paraformaldehyde decreased adherence (** P ,
0.001) to EC treated with XO/HX. Values are means 6 SE of 6
individual determinations.
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Fig. 8. Incorporation of [3H]acetate into platelet-activating factor
(PAF) increased (* P , 0.001) after treatment of EC with XO (10
mU/ml) and HX (200 µM), compared with untreated EC. Catalase
(815 U/ml) decreased (** P , 0.01) [3H]acetate incorporation in
XO/HX-treated EC. SOD (30 U/ml) did not alter [3H]acetate incorporation in XO/HX-treated EC (P . 0.05). Values are means 6 SE of 6
individual determinations. cpm, Counts/min.
Fig. 10. Neutrophil adherence increased (* P , 0.001) after treatment of EC with XO (10 mU/ml) and HX (200 µM). Treatment of EC
and neutrophils with antibodies against intercellular adhesion molecule-1 (ICAM-1; aCD54) or CD11b (4 µg/ml) decreased (** P , 0.001)
neutrophil adherence to XO/HX-treated EC. Treatment of EC and
neutrophils with irrelevant isotype antibodies or antibodies against
CD11a did not affect neutrophil adherence (P . 0.05). Values are
means 6 SE of 6–12 individual determinations. IgG, immunoglobulin G.
XANTHINE OXIDASE AND NEUTROPHIL ADHERENCE
871
cellular, XO is likely the source of oxygen metabolites.
In the present situation with exogenous XO, the diffusion of superoxide anion radical into EC from external
sites through anion channels may limit its participation, or the specific targets may differ.
We also found evidence for PAF as an EC-derived
intermediate in neutrophil adherence. First, incorporation of [3H]acetate into PAF increased after stimulation
Fig. 12. Neutrophil adherence increased (* P , 0.001) after treatment of EC with XO (10 mU/ml) and HX (200 µM). Treatment of EC
with antibodies to P-selectin (a-CD62) did not (P . 0.05) decrease
neutrophil adherence to XO-treated EC. Values are means 6 SE of 6
individual determinations.
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in concentrations we have previously found in plasma
to affect lung neutrophil retention in vivo (33). The
effect was rapid, occurring within 10 min, and did not
require de novo protein synthesis, precluding involvement of nonconstitutive EC ligands such as E-selectin.
Curiously, neutrophil adherence decreased to baseline
levels by 40 min. Although the reason for this timedependent decrease in adherence is not clear, it may
relate to a decrease in EC PAF levels, as occurs after
stimulation of EC with bradykinin (35) or thrombin
(28). Alternatively, b2-integrin expression may also
decrease despite continued neutrophil stimulation (24).
The effect of XO-derived oxidants appeared to be
primarily on EC, since treatment of EC, but not neutrophils, increased adherence. We did not find evidence for
formation of plasma-derived factors to mediate this
effect. The direct effect of XO on EC is particularly
relevant, since XO binds to anionic EC surface moieties
both in vitro and in vivo in a heparin-reversible manner
(1, 31). Our data are consistent with these prior studies
and further suggest that EC-bound XO can activate
neutrophil adherence mechanisms. This close approximation of XO to EC may be particularly germane in
vivo, as a means of bypassing the antioxidant-rich
milieu of blood. We found evidence for this effect when
EC were exposed to XO in 50% plasma. Human plasma
is rich in oxidant-scavenging activity (20) and could
potentially dampen the effect of XO on EC. In 50%
plasma, however, XO continued to mediate an increase
in neutrophil adherence, consistent with a site-specific
mode of action of XO at the cell surface. In this
environment, as opposed to HBSS, heparin decreased
neutrophil adherence when added concurrently with
XO and HX, providing further evidence that displacement of XO from the surface of EC allows plasma
scavengers to intervene and diminish the effects of
delocalized XO.
H2O2 was primarily involved in mediating static
neutrophil adherence, since catalase, but not SOD or
the membrane-permeable SOD mimic Tempol, decreased adherence. It is not clear why some studies of
reperfusion implicate XO-derived superoxide anion radical rather than H2O2 in mediating adherence (10, 34),
although in such studies endogenous, presumably intra-
Fig. 13. A: treatment of EC with XO (10 mU/ml) and HX (200 µM)
increased no. of rolling neutrophils (* P , 0.001) at all 3 shear rates.
Treatment of EC with antibodies against ICAM-1 decreased no. of
rolling neutrophils at lowest shear rate (** P , 0.05). Treatment of
EC with antibodies against P-selectin (a-CD62) decreased (** P ,
0.05) no. of neutrophils rolling on XO-treated EC at all 3 shear rates.
Treatment of EC with antibodies against both P-selectin and ICAM-1
(a-CD54) further decreased (# P , 0.001) no. of neutrophils rolling on
XO-treated EC. B: treatment of EC with XO and HX decreased (* P ,
0.01) neutrophil rolling velocity only at a shear rate of 38 s21.
Treatment of EC with antibodies to P-selectin and/or ICAM-1 did not
significantly affect rolling velocity (P . 0.05). C: treatment of EC with
XO and HX increased (* P , 0.01) no. of firmly adherent neutrophils
at all 3 shear rates. Treatment with antibodies against P-selectin
decreased (** P , 0.01) firm adherence to XO-treated EC at 60 and 96
s21. Treatment with antibodies against ICAM-1 decreased (** P ,
0.05) firm adherence to XO-treated EC at all 3 shear rates. Treatment
with antibodies to both P-selectin and ICAM-1 decreased (# P , 0.001)
adherence to XO-treated EC at all 3 shear rates.
872
XANTHINE OXIDASE AND NEUTROPHIL ADHERENCE
pears to be P-selectin, whose expression increased after
exposure to XO. A dynamic system was required to
demonstrate an effect of the monoclonal antibody
against P-selectin, suggesting that the integrin-mediated adherence dominated the effect seen in the static
assay. This may explain the apparent discrepancy
between studies that have investigated the role of
P-selectin using static (29) vs. dynamic (9) systems. The
cooperativity between the selectin and integrin adherence systems was more evident at higher shear rates,
where interference with P-selectin markedly suppressed firm adherence. This is consistent with the
notion that, under higher levels of shear, rolling is a
prerequisite for firm adherence (19). In contrast, XOmediated firm adherence was not significantly reduced
by the antibody to P-selectin at the lowest shear rate
tested. Indeed, observations in vivo suggest that ICAM1-mediated firm adherence can occur at low shear rates
even with effective blockade of P-selectin sites (16). A
potential limitation of this study is the use of bovine
rather than human EC. However, our data suggest that
human neutrophils appear capable of interacting with
bovine EC via both integrin and selectin ligand pairs.
In summary, extracellular XO can associate closely
with EC and stimulate adherence of neutrophils through
interactions with endothelial ICAM-1 and P-selectin.
We suggest that circulating XO may contribute to the
initial recruitment of neutrophils to secondarily affected vascular beds.
This work was supported by the American Heart Association and
National Heart, Lung, and Blood Institute Grants R29-HL-52591,
R01-HL-5582, and P50-HL-0784. L. S. Terada is an Established
Investigator of the American Heart Association, and B. M. Hybertson
is a fellow of the Parker B. Francis Foundation.
Address for reprint requests: L. S. Terada, Univ. of Colorado
Health Sciences Center, Box C322, 4200 E. Ninth Ave., Denver, CO
80262.
Received 2 April 1996; accepted in final form 20 September 1996.
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