Department of Physiology, University of Western Ontario, London, N6A 5C1; and Vascular Biology Program, Lawson Health Research Institute, London, Ontario, Canada, N6A 4G5
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ABSTRACT |
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In vitro, nitric oxide (NO) decreases
leukocyte adhesion to endothelium by attenuating endothelial adhesion
molecule expression. In vivo, lipopolysaccharide-induced leukocyte
rolling and adhesion was greater in inducible NO synthase (iNOS)/
mice than in wild-type mice. The objective of this study was to assess
E- and P-selectin expression in the microvasculature of iNOS
/
and
wild-type mice subjected to acute peritonitis by cecal ligation and
perforation (CLP). E- and P-selectin expression were increased in
various organs within the peritoneum of wild-type animals after CLP.
This CLP-induced upregulation of E- and P-selectin was substantially reduced in iNOS
/
mice. Tissue myeloperoxidase (MPO) activity was
increased to a greater extent in the gut of wild-type than in iNOS
/
mice subjected to CLP. In the lung, the reduced expression of
E-selectin in iNOS
/
mice was not associated with a decrease in MPO.
Our findings indicate that NO derived from iNOS plays an important role
in sepsis-induced increase in selectin expression in the systemic and
pulmonary circulation. However, in iNOS
/
mice, sepsis-induced
leukocyte accumulation is affected in the gut but not in the lungs.
neutrophils; cecal ligation and perforation; myeloperoxidase activity; intracellular adhesion molecule-2
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INTRODUCTION |
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THE RECRUITMENT OF
LEUKOCYTES to the endothelial cell surface of the
microvasculature, and subsequent infiltration into the interstitium, is
believed to be the initial event that leads to multiple organ
inflammation and dysfunction during sepsis (16, 24). This
process involves a series of well-coordinated adhesive interactions
mediated by adhesion glycoproteins expressed on leukocytes and the
endothelium. The tethering and rolling of neutrophils (PMN)
along the endothelium are regulated by the selectins (L-selectin on PMN
and P- and E-selectin on endothelium), whereas firm adhesion is
mediated primarily by intercellular adhesion molecule
(ICAM)-1/2-integrin interactions (24). Once
firmly adherent to the endothelium, the PMN flatten, extend pseudopodia
between endothelial cells, and emigrate into the interstitium. These
invading PMN are believed to cause damage to the tissue
(23).
PMN rolling along the endothelium is a prerequisite for subsequent
adhesion and emigration. The endothelial cell selectins are not
constitutively expressed on the cell surface. P-selectin, which is
stored in the Weible-Palade bodies of endothelial cells, is rapidly
expressed (within minutes) on the endothelial cell surface after
stimulation (e.g., histamine, oxidants). There is also a
transcription-dependent mechanism that can upregulate the expression of
P-selectin (8, 9). It is believed that the stored
P-selectin is responsible for the early recruitment of leukocytes,
whereas the transcription-dependent expression of P-selectin is
involved in the leukocyte rolling that is observed several hours after
the onset of inflammation (8, 9). Although there is no
preformed pool of E-selectin, endotoxin [e.g., lipopolysaccharide (LPS)] and cytokines [e.g., tumor necrosis factor- and
interleukin-1
] are capable of stimulating the synthesis and
expression of E-selectin on the surface of endothelial cells (10,
15). The critical role of selectins in inflammation is evidenced
by studies that show that interfering with selectin-mediated rolling of
PMN prevents PMN infiltration and organ dysfunction (3, 4, 13,
17, 22).
Nitric oxide (NO), synthesized by NO synthase (NOS), is believed to
modulate leukocyte-endothelial cell interactions by acting as an
endogenous antiadhesive molecule (12). In vitro studies indicate that NO can attenuate endothelial cell adhesion molecule expression [E-selectin, ICAM-1, and vascular cell adhesion
molecule-1] (6, 27, 28). The functional result of this
inhibition is reduced PMN adhesion to endothelial cells (5, 11,
21). The constitutive isoform of NOS (cNOS) is continually
expressed and produces small fluxes of NO, whereas the inducible NOS
(iNOS) is a high-output isoform. In general, during sepsis, the
activity of cNOS is decreased and iNOS activity is increased, resulting in substantially elevated levels of NO production (16). In
the present study, we assessed the role of NO (derived from iNOS) in
the expression of E- and P-selectin in mice exposed to sepsis. Our
approach to address this issue involved the use of iNOS-deficient (iNOS/
) mice.
We have recently reported that the induction of peritonitis in mice by
cecal ligation and perforation (CLP) results in symptoms that closely
mimic those observed clinically (hypotension, neutropenia, elevated
blood lactate). In addition, CLP-induced peritonitis in these animals
leads to increased expression of endothelial selectins in various
organs (1). The purpose of the present study was
to use our acute model of murine polymicrobial sepsis to determine the
role of iNOS in the early (within 6 h) upregulation of the
selectins on endothelial cells. As such, this is the first study to
quantify the level of expression of E- and P-selectin in iNOS/
mice
following a septic insult. In addition, we present evidence that NO
derived from iNOS contributes to selectin expression on the vascular
endothelium during sepsis.
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METHODS |
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Animal protocol.
Male C57/BL6 mice and iNOS/
mice (C57/BL6 background) weighing
20-30 g were obtained from Charles River Canada (St. Constant, PQ)
and Jackson Laboratories (Bar Harbor, ME), respectively. Sepsis was
induced in mice using the CLP model as previously described (1,
20). Briefly, animals were anesthetized with 150 mg/kg body wt
of ketamine and 7.5 mg/kg sc of xylazine. A midline incision ~2 cm
long was made in the abdomen to expose the cecum and adjoining intestine. With the use of 2-0 silk, a ligature was placed around the
cecum immediately distal to the ileocecal valve. The cecum was then
opened by making a 5-mm incision at the antimesenteric border. The
laparotomy was then closed using 4-0 silk, and the animal was given a 1 ml subcutaneous injection of saline for fluid resuscitation.
Sham-operated mice underwent laparotomy but no ligation or cecal
perforation. Control animals did not undergo surgery.
Systemic variables.
Six hours after induction of CLP, mean arterial pressure (MAP) was
measured from a carotid arterial catheter that was connected to a
pressure transducer and recorded with a multichannel amplifier recording system (Hewlett Packard 78353A). Blood samples were taken for
analysis of both arterial blood levels of lactate and metabolic end
products of NO (NOx).
NOx chemiluminescence detection.
The metabolic end products of NO production (NO2
and
NO3
; NOx
) were determined
(29) in plasma samples using chemiluminescence detection
in a Sievers Model 270 B analyzer (Sievers Instruments, Boulder, CO)
with a Shimadzu Chromatopac C-R1A integrator (Shimadzu, Kyoto, Japan).
Briefly, an aliquot of plasma was injected into a glass purge vessel
containing a saturated solution of vanadium (III) chloride in
hydrochloric acid (1 M) at 90°C, resulting in reduction of both
NO2
and NO3
to NO. This gas-phase
NO is carried by a continuous stream of inert gas (helium) from the
purge vessel into an ozone-containing reaction chamber in the NO
analyzer. The resultant chemiluminescent reaction between NO and ozone
is detected by a photomultiplier tube, yielding an electric signal (mV)
that was analyzed for area under the curve (AUC) calculations on the
chromatographic integrator. The analyzer was calibrated daily and
rechecked periodically during analysis of samples, which were
referenced to a standard curve of AUC (mV × s) vs.
NO3
concentration (50 nM-500 µM;
r2 > 0.999).
Adhesion molecule expression.
E- and P-selectin expression on the vascular endothelium of various
organs was determined by using the dual radiolabeled antibody technique
(1, 2, 9). The monoclonal antibodies (MAbs) used for the
in vivo assessment of E-selectin expression were 10E9.6 (PharMingen), a
binding rat IgG2a against mouse E-selectin and R35-95
(PharMingen), and a nonbinding purified rat IgG2a, isotype-matched
negative control. The P-selectin MAbs used were RB40.34 (PharMingen), a
binding rat IgGg1 against mouse P-selectin, and the same nonbinding rat
IgG2a,
described above. The use of both binding and nonbinding MAbs
simultaneously allows for the correction of any nonspecific
accumulation of the binding MAbs in a given tissue. The determination
of ICAM-2 expression was made using a purified rat anti-mouse CD102 MAb
(3C4; PharMingen).
MPO activity.
Tissue samples were weighed, frozen, and analyzed for determination of
MPO activity as previously described (2). MPO activity is
commonly used as a measure of PMN accumulation in tissues. Briefly,
samples were thawed, suspended (10% wt/vol) in potassium phosphate
buffer (KPi; 50 mM, pH 6.0) containing 0.5%
hexadecyltrimethylammonium bromide (Sigma), and homogenized. One
milliliter of homogenate was sonicated three times and spun at 20,000 g for 10 min (4°C). The reaction was started by incubating
100 µl of supernatant with O-dianisidine solution (30 µl
of 20 mg/ml O-dianisidine, 2,900 µl of 50 mM
KPi, 30 µl of 20 mM H2O2) for 5 min at 25°C. The reaction was stopped by adding 30 µl of 2% sodium
azide. The change in absorbency was read after 5 min at 450 nm using a
microplate reader (Bio-Rad Model 3550-UV). MPO activity was expressed
as the amount of enzyme necessary to produce a change in absorbency of
1.0 · min1 · g wet wt
1.
Statistics.
Comparison between genotypes (wild-type vs. iNOS/
) of mice after
CLP was made using one-way ANOVA followed by Student's t-test (with Bonferroni correction for multiple comparisons)
and within a given genotype using a two-tailed t-test.
Statistical significance for all tests was set at P < 0.05.
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RESULTS |
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As shown in Table 1, 6 h after
CLP there was a significant reduction in MAP compared with sham animals
for both wild-type and iNOS/
mice. MAP was reduced to similar
levels in both wild-type and iNOS
/
mice. Furthermore, animals
subjected to CLP also had an elevated level of circulating lactate,
which is indicative of the development of sepsis. These changes in
systemic variables are consistent with our previous studies using a
similar model of sepsis in mice (1). All animals survived
the 6-h CLP procedure.
|
The induction of CLP in wild-type mice resulted in a significant
elevation in plasma NOx levels, a measure of the
metabolic end products of NO production (Fig.
1). In contrast, there were negligible
levels of NO2
and NO3
in both CLP
and sham-operated iNOS
/
mice.
|
Expression of E-selectin 6 h after CLP was significantly increased
in organs within the peritoneum (i.e., small and large bowel, pancreas)
and an organ outside of the peritoneum (i.e., lung) in wild-type
animals (Fig. 2). In iNOS/
mice,
however, there was no increase in the expression of E-selectin in these organs after CLP compared with sham. The data for other organs examined
are summarized in Table 2. In general,
similar trends were noted in other organs within the abdominal cavity.
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|
In terms of P-selectin expression, variable responses were noted for
both wild-type and iNOS/
mice. Significant increases in P-selectin
were noted 6 h after CLP in wild-type mice in organs within the
abdominal cavity (small and large bowel, pancreas), but not in the lung
(Fig. 3). There were significant
decreases in the level of expression of P-selectin in both the large
bowel and pancreas of iNOS
/
mice following CLP (Fig. 3). The data for other organs examined are summarized in Table
3. In general, similar results were noted
for organs in the abdominal cavity.
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ICAM-2 expression in CLP wild-type and iNOS-deficient mice was assessed
to determine tissue perfusion (Table 4).
It has previously been reported that ICAM-2 is constitutively expressed
on murine endothelial cells, and expression is not increased by
stimulation with inflammatory cytokines (14, 31).
Consistent with these findings, it is evident from Table 4 that, in
general, there were no major changes in the expression of ICAM-2 after
CLP in both wild-type and iNOS-deficient mice. These findings would
indicate that, despite the hypotensive state of the septic animals,
tissue perfusion was adequate for quantification of adhesion molecule expression.
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To determine whether these variations in the level of expression of E-
and P-selectin correlated with changes in the accumulation of
leukocytes in these organs, MPO activity in representative organs,
i.e., the small bowel and lung, was assessed. As shown in Fig.
4, after the induction of CLP, MPO
activity was increased in the small bowel of wild-type mice compared
with sham animals. In contrast, induction of peritonitis in iNOS/
mice did not result in an increase in MPO activity in the small bowel.
CLP induced significant increases in lung MPO activity in both
wild-type and iNOS
/
mice.
|
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DISCUSSION |
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The purpose of the present study was to assess the role of iNOS
activity in the in vivo regulation of selectin expression after
induction of sepsis (CLP) by using iNOS/
mice. To confirm that
these mice had not compensated for the lack of the iNOS gene, possibly
by increased cNOS activity, we measured plasma NOx
levels (Fig. 1). Six hours after CLP, NOx
levels were
significantly elevated in wild-type mice, indicative of increased iNOS
activity and subsequent NO production. More importantly, our data
indicate that there was no compensation for the lack of iNOS activity,
because NOx
levels in the iNOS
/
mice were well
below those of the wild-type mice, an observation consistent with
previous studies (11, 18).
As previously reported (1), we show that 6 h after of
CLP, E- and P-selectin expression is elevated within the
microvasculature of organs in the abdominal cavity of wild-type mice.
This CLP-induced increase in selectin expression is substantially
reduced in iNOS/
mice (Figs. 2 and 3). Moreover, this reduced
expression corresponds to a reduced level of PMN recruitment in a
representative organ, i.e., small bowel (Fig. 4). Together, these
observations provide a compelling argument against the currently held
view that NO inhibits adhesion molecule expression and PMN infiltration.
In apparent contrast to our observations, previous studies using
iNOS/
animals have shown more pronounced leukocyte-endothelial cell
interactions (rolling) after endotoxin challenge (11). One
possible explanation for these seemingly disparate results is that the
previous study relied on LPS as the challenge, which may not reflect
the complex situation that exists during polymicrobial sepsis. In
support of this possibility are the results of a recent study in which
we measured E- and P-selectin expression 6 h after induction of
CLP in LPS-sensitive (C3Heb/FeJ) and LPS-insensitive (C3H/HeJ) mice
(1). The CLP-induced increase in selectin expression was
the same in both the LPS-sensitive and -insensitive mice. These
findings indicate that bacteria-derived endotoxin (LPS) does not play
an important role in the CLP-induced increase in selectin expression.
Thus some degree of caution should be used in relating LPS-induced
inflammatory responses to those induced by polymicrobial sepsis.
Our data indicate that NO production is important for the expression of
E- and P-selectin after CLP. However, in vitro studies (6,
26-28) have found that NO can attenuate endothelial cell adhesion molecule expression. Two major differences between these latter studies and our present study exist that might offer an explanation for this discrepancy. First, the previous studies employed
either NO donors or inhibitors to examine the effect of NO production
on adhesion molecule expression in cultured human endothelial cells. In
our study, we assessed the in vivo expression of selectins on
endothelium of various organs in genetically altered mice. Thus it is
conceivable that in situ endothelial cells of iNOS/
animals respond
differently from isolated cultured human cells. Second, adhesion
molecule expression was induced in vitro by stimulation of endothelial
cells with either interleukin-1 or tumor necrosis factor-
. This
approach may not be representative of the in vivo situation, in which
multiple factors are involved in endothelial cell stimulation during
polymicrobial sepsis. Irrespective of the explanation for these
seemingly dichotomous observations, a recent study (25)
using a different model of inducing tissue inflammation in vivo lends
credence to our findings. The ischemia/reperfusion-induced increase in
E-selectin expression in the small intestine of wild-type mice was
completely abolished in iNOS
/
animals. Given this situation, further studies are warranted to address the role of NO in adhesion molecule expression in vivo and in vitro to attempt to reconcile this controversy.
The CLP-induced E- and P-selectin expression was diminished in organs
located within the peritoneum of iNOS/
mice. This decreased
expression of endothelial selectins was associated with a reduction in
small intestinal MPO activity, indicating that selectins play an
important role in PMN recruitment to the gut. In contrast, E-selectin
expression on lung endothelium was reduced and P-selectin expression
was unaffected in iNOS
/
mice (Fig. 4). However, MPO accumulation in
the lungs of iNOS
/
mice was the same as in their wild-type
counterparts. These findings suggest that either 1)
P-selectin is sufficient for PMN accumulation in the lung or
2) leukocyte sequestration in the lung is independent of
selectin expression. These results can be explained by organ-specific recruitment of leukocytes. Previous studies have indicated that blockade of selectin (or integrin) function during CLP results in
reduced peritoneal accumulation of PMN, whereas lung PMN sequestration is unaffected (19, 30). Thus our data are consistent with the current consensus that in some organs (i.e., the small intestine) the classic adhesion pathway may predominate, whereas in the lung, PMN
may adhere to pulmonary capillaries (capillary plugging) independently of adhesion molecule function (7).
In conclusion, the results of the present study indicate that the
production of NO by iNOS plays a critical role in the expression of E-
and P-selectin during polymicrobial sepsis in vivo. Specifically, the
CLP-induced E- and P-selectin expression on endothelial cells is
reduced in organs located within the abdominal cavity of iNOS/
mice
compared with their wild types. This reduction of endothelial selectin
expression is associated with decreased PMN accumulation in the gut.
Together, our findings indicate that NO plays an important role in
modulating peritonitis-induced endothelial cell adhesion molecule
expression and leukocyte sequestration in organ systems within the peritoneum.
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ACKNOWLEDGEMENTS |
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We thank Drs. D. N. Granger and P. Bauer for invaluable
assistance in establishing the dual radiolabeled antibody technique in
the laboratory. We thank R. Bateman for technical assistance with the
NOx chemiluminescence detection.
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FOOTNOTES |
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This work was supported by Grants MT-13940 and GR-12816 from the Canadian Institutes of Health Research (CIHR). C. Lush is supported by the Doctoral Research Award from the CIHR.
Address for reprint requests and other correspondence: P. R. Kvietys, Lawson Health Research Institute, Vascular Biology Program, 375 South St., Rm. C210, London, ON, Canada N6A 4G5 (E-mail: pkvietys{at}julian.uwo.ca).
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.
Received 22 March 2000; accepted in final form 25 August 2000.
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