1 Hepatic Hemodynamic Laboratory, Liver Unit, Institut Malalties Digestives, Hospital Clinic, and 2 Immunology Unit, Department of Cellular Biology and Pathology, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Medical School, University of Barcelona, 08036 Barcelona, Spain
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ABSTRACT |
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Altered leukocyte/cytokine response to inflammation has been observed in human and experimental portal hypertension. The aim of this study was to characterize leukocyte adhesion in portal hypertensive (PPVL) rats stimulated with endotoxin. Leukocyte rolling, adhesion, and migration assessed by intravital microscopy were impaired in mesenteric venules after lipopolysaccharide administration (150 µg/kg) in PPVL vs. sham-operated rats. Analysis of leukocyte L-selectin expression and soluble L-selectin showed that this defective adhesion was related to increased L-selectin shedding. In vitro experiments using isolated leukocytes treated with phorbol 12-myristate 13-acetate showed that monocytes and neutrophils but not lymphocytes were hyperreactive to cell activation, as measured by CD11b overexpression and increased L-selectin shedding in PPVL rats. However, neutrophil emigration in liver sinusoids and in the lung 3 h after endotoxin injection were similar in both groups of animals. Thus the alterations in leukocyte activation and adhesion molecule expression observed in this study may contribute to a better understanding of the higher susceptibility and severity of bacterial infections in cirrhotic patients with portal hypertension.
portal hypertension; sepsis; L-selectin; cirrhosis; adhesion molecules
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INTRODUCTION |
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IN THE NORMAL COURSE OF
AN infection, the immune system acts to protect the host from
pathogenic infections. Many bacteria induce local inflammatory
processes on entry. If infective agents or bacterial products, such as
endotoxin, spread to the bloodstream, the same mechanisms whereby the
immune system so efficiently contains local infection instead become
damaging to the host. A systemic release of inflammatory mediators,
such as tumor necrosis factor (TNF-) and interleukin (IL)-1, induces
leukocyte infiltration and inflammation in various organs and
apoptosis of the hepatocytes and causes septic shock (23,
32, 33).
Bacterial infections by Gram-negative organisms are frequent in
patients with cirrhosis and portal hypertension, are often severe, and
may lead to septic shock and death (17, 23). There is
evidence suggesting that leukocyte and cytokine responses to endotoxin
(lipopolysaccharide, LPS) or proinflammatory factors are altered in
cirrhotic patients and portal hypertensive animals. Patients
with chronic liver disease respond to sepsis with a greater and
long-lasting increase in the plasma levels of TNF- and IL-6 (1). A positive correlation between the levels of TNF-
and IL-6 and mortality in patients with alcoholic hepatitis and
cirrhosis has also been reported (8, 19, 25). Furthermore,
cultured monocytes from cirrhotic patients have an increased production of TNF-
in response to endotoxin (4). We have shown
that portal hypertensive rats have enhanced TNF-
plasma levels after
LPS injection, together with a significant upregulation of CD11b/CD18 expression on monocytes (22). In addition, elevated levels
of TNF-
have been shown to be related to an overexpression of the intercellular adhesion molecule (ICAM)-1, a ligand of leukocyte integrin CD11b/CD18, on the endothelium of portal hypertensive rats
after LPS injection (21). Because this pair of
adhesion molecules mediate firm adhesion and subsequent emigration of
leukocytes from the blood to the inflamed endothelium, leukocyte
emigration could be increased in these animals. Moreover, these
intensified responses to LPS in portal hypertensive rats are
accompanied by evidence of liver damage, as shown by a marked increase
in serum transaminases (22). Paradoxically, portal
hypertensive rats have markedly reduced leukocyte rolling, adhesion,
and migration in mesenteric venules in response to the lipid
proinflammatory mediators platelet-activating factor (PAF) and
leukotriene B4 (LTB4). This observation
indicates that portal hypertension could be associated with a defective
inflammatory response (20).
Therefore the aim of this study was to characterize leukocyte inflammatory response in portal hypertensive rats stimulated with endotoxin. Intravital microscopy was used to assess endotoxin-induced leukocyte rolling, adhesion, and migration in mesenteric venules in an animal model of portal hypertension. The expression of L-selectin, an adhesion molecule that mediates leukocyte rolling (10, 31, 34), and its soluble form were also examined. In vitro experiments were performed to study the response of isolated leukocytes from these animals to cell activation. Because neutrophil accumulation has been shown to cause endotoxin-induced liver and lung injury (12), sinusoidal hepatic neutrophil sequestration and lung myeloperoxidase (MPO) activity were also evaluated.
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MATERIALS AND METHODS |
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Portal hypertensive rats. Male Sprague-Dawley rats (weight 250-300 g) were obtained from Charles River (Saint Aubin-lès-Elbeuf, France). Portal hypertension was induced under ketamine anesthesia (Ketalar, 100 mg/kg body wt im; Parke-Davis, Morris Klein, NJ) by partial portal vein ligation (PPVL) as previously described (24). In brief, the portal vein was isolated and a calibrated constriction was performed using a single ligature of 3-0 silk around the portal vein and a 20-gauge blunt-tipped needle, which was later removed. The surgical procedure was performed in sterile conditions. In sham-operated controls the portal vein was isolated but not ligated. Animals were allowed to recover and had free access to water and food until the day of the study.
All experiments were performed according to the criteria of the Committee for the Care and Use of Laboratory Animals in the Hospital Clínic i Provincial and Institut d'Investigacions Biomèdiques August Pi i Sunyer.Hemodynamic measurements. Sham-operated and PPVL rats were studied 15 days after surgery, when portal hypertension had fully developed (24). Sham-operated (n = 8) and PPVL (n = 8) animals were anesthetized with ketamine, and polyethylene (PE)-50 catheters (Portex, Técnicas Médicas, Barcelona, Spain) were introduced in the femoral vein to obtain blood samples and in the carotid artery to monitor mean arterial pressure. To measure portal pressure, the portal vein was also cannulated through an ileocolic vein with a PE-50 catheter. After verification that free reflux of blood was obtained, the catheter was fixed to the mesentery by applying a drop of cyanoacrylate glue, and the abdomen was closed with surgical sutures. All catheters were connected to highly sensitive pressure transducers that were calibrated before each study, and blood pressure values were registered on a multichannel recorder (Lectromed MT6-PX; St. Peters, Jersey, Channel Islands) (24). Measurements were taken at rectal temperature 37 ± 0.5°C. Whole blood was then collected from the femoral vein or from the inferior vena cava in heparinized tubes and centrifuged at 4°C, and the plasma obtained was quickly frozen.
Intravital microscopy in mesenteric venules.
Rats were anesthetized with thiobutabarbital (100 mg/kg ip; Research
Biochemicals International, Natick, MA). The trachea and the right
carotid artery were cannulated, and arterial blood pressure was
recorded. A midline abdominal incision was made using a thermocauter to
prevent local bleeding, and part of the mesentery from the small bowel
was exteriorized. Rats were placed over a steel microscope board in a
supine position, and the section of the mesentery was observed through
a glass slide covering a 3.5 × 3.5-cm hole centered on the board.
The exposed intestine was covered with a bicarbonate buffered saline
(BBS, pH 7.4)-soaked gauze, and the mesentery was continuously
superfused with BBS at 37°C and a flow rate of 2.5 ml/min to avoid
dehydration. The superfusate temperature was maintained by pumping the
solution through a heat exchanger warmed with a constant- temperature
circulator (Haake, Berlin, Germany). The steel board was placed on the
stage of an inverted microscope (Diaphot 300; Nikon, Tokyo, Japan)
equipped with a CF Fluor ×40 objective lens (Nikon). The preparation
was transilluminated with a 12-V, 100-W, direct current stabilized light source. A three-charged coupled device camera (model
DXC-930P; Sony, Tokyo, Japan), mounted on the microscope, projected the image onto a color monitor (Triniton KX-14CP1, Sony), and the images
were recorded on video tape (SR-S368E videocassette recorder; JVC,
Tokyo, Japan). Time and date were superimposed on both taped and live
images with a date-time generator (KPM Systems, Barcelona, Spain). Single unbranched mesenteric venules between 25 and 35 µm in diameter and >100 µm in length were selected for study. Venular diameter (Dv) was measured online using
a video caliper (Microcirculation Research Institute, Texas A & M
University, College Station, TX). The number of rolling, adherent, and
migrated leukocytes was measured offline during playback analysis of
videotaped images. A leukocyte was considered adherent to venular
endothelium if it remained stationary for a period 30 s.
Adherent leukocytes were quantified as the number per 100-µm length
of vessel. Leukocyte emigration was expressed as the number of
leukocytes per microscopic field (1.7 × 10
2
mm2). Rolling leukocytes were defined as those white blood
cells moving at a slower velocity than that of erythrocytes within a given vessel. Leukocyte rolling velocity (VWBC)
was determined from the time required for a leukocyte to traverse a
50-µm distance along the length of the venule. Flux of rolling
leukocytes was measured by counting white blood cells seen rolling past
a defined reference point within the 100-µm vessel length using the
same reference point throughout the experiment. Centerline red blood cell velocity (VRBC) was also measured online
using an optical Doppler velocimeter (Microcirculation Research
Institute). Venular blood flow was calculated, assuming cylindrical
geometry, from the product of mean VRBC
(Vmean = VRBC/1.6)
and microvascular cross-sectional area. Venular wall shear rate (
)
was calculated from the Newtonian definition:
= 8 (Vmean/Dv).
Experimental protocol. Sham-operated (n = 15) and PPVL (n = 15) animals were treated intraperitoneally with 150 µg/kg of LPS or PBS. Animals were studied 0, 1, or 3 h after LPS injection. Part of the mesentery was placed on the microscope board, and two to five single unbranched venules were selected for study. The mesentery was continuously superfused with BBS at 37°C to avoid dehydration. After a brief stabilization period, images from the mesenteric preparation were recorded on videotape for 5 min.
Neutrophil emigration in liver sinusoids. Formalin-fixed portions of the liver were paraffin embedded, and 5-µm-thick sections were cut and stained with hematoxylin and eosin. The number of neutrophils present within the sinusoids was counted. Five rats were used for each group. For the analysis of sinusoidal leukocyte migration, five microscopic fields at a magnification of ×400 were randomly chosen from each rat, and all of the sinusoids included in the field were analyzed.
Determination of soluble L-selectin.
A sandwich ELISA was developed to detect the soluble form of rat
L-selectin. The anti-rat L-selectin monoclonal antibodies (MAb) LAM
1.116 (28) diluted to 3 µg/ml was used as the capture antibody. The presence of soluble L-selectin was detected
using biotinylated anti-HRL2 MAb at 3 µg/ml (Pharmigen, San Diego,
CA) (30) and avidin-horse radish peroxidase with
o-phenylenediamine (0.125% wt/vol, Sigma, St. Louis, MO) as
substrate. Rat plasma samples were run in triplicate and diluted at
1:10 to obtain a measurement in the linear range of the assay.
Absorbances at 450 nm were measured by using an MRX Microplate ELISA
reader (Dynatech, Denkendorf, Germany). The concentration of soluble
L-selectin was determined by using a standard curve constructed for
each ELISA plate with reference plasma produced from a pool obtained from control rats. The soluble L-selectin level in the reference plasma
was assigned as 1,000. Results are expressed as relative units (U),
interpolating the values in each standard curve. The sensitivity of the
assay was 12.5 U, which allows the detection of soluble L-selectin in
the plasma of control rats. Therefore, the method used for this study
is more sensitive than the one previously described, which was not able
to detect soluble L-selectin in normal sera (30).
Determination of TNF- levels.
TNF-
levels were determined using a sensitive ELISA kit (Genzyme)
specific for rat TNF-
, following the manufacturer's instructions. The detection limits were 10 and 2,240 pg/ml, and results were expressed as pg/ml. The absorbances were measured at 450 nm using an
MRX Microplate ELISA reader (Dynatech).
Experimental protocol.
Sham-operated (n = 8) and PPVL (n = 8)
animals were treated intravenously with LPS (Escherichia
coli serotype 011:B4, endotoxin, Sigma). The animals were divided
into two groups, which received either 150 µg/kg of endotoxin or
vehicle (PBS). Whole blood was collected from the jugular vein 30, 60, 120, and 180 min after LPS injection. The samples were centrifuged at
4°C, and the plasma obtained was quickly frozen. Plasma samples were
used to determine rat soluble L-selectin and TNF- levels.
Indirect immunofluorescence assay. Indirect immunofluorescence assays for L-selectin and CD11b surface expression on leukocytes were performed using biotinylated MAb LAM 1.116 (28) and CD11b PE (clone MCROX-42; Serotec, Oxford, UK) by double fluorescence. Aliquots of whole blood (50 µl) were washed twice in PBS. Biotinylated MAb was detected by a 30-min incubation with FITC-conjugated avidin (Sigma) at 1:100 dilution. Erythrocytes were lysed using the Ortho Lysing Reagent (Ortho Diagnostic Systems, Raritan, NJ) as detailed by the manufacturer. FITC and PE fluorescence were measured on a FACSCalibur Flow Cytometer (Becton Dickinson Immunocytometry Systems, San José, CA). Neutrophils, monocytes, and lymphocytes were identified for analysis by forward- and right-angle light scatter, and gates were set to exclude other cell types. Routinely, 10,000 cells were analyzed. The data are presented as mean channel fluorescence (4 decade log scale). Changes in L-selectin shedding are calculated as the percentage of decreased expression over baseline conditions.
In vitro stimulation assays. Whole blood from PPVL (n = 6) and sham-operated animals (n = 6) was taken 15 days after surgery. Aliquots of whole blood (50 µl), previously washed in PBS, were incubated with different doses of phorbol 12-myristate 13-acetate (PMA, 0.1-100 ng/ml, Sigma) for 15 min at 37°C. After centrifuging and decanting the supernatant, indirect immunofluorescence assays were performed with biotinylated L-selectin and CD11b PE to measure CD11b and L-selectin surface expression on leukocytes.
MPO measurements.
Lung MPO activity was assayed spectrophotometrically using
o-dianisidine as substrate. Rats were killed 3 h after
LPS injection (150 µg/kg). The lung was frozen at 80°C
immediately after the tissue was rinsed with ice-cold saline. Lung
tissue was homogenized for 30 s in 4 ml 20 mmol/l potassium PBS
(pH 7.4), and centrifuged for 20 min at 20,000 g at 4°C.
The pellet was resuspended in 4 ml of 50 mmol/l potassium PBS (pH 6.0)
containing 0.5 g/dl hexadecyltrimethylammonium bromide. Samples were
sonicated for 90 s at full power, incubated in a 60°C water bath
for 2 h, and centrifuged 10 min at 20,000 g. One
hundred microliters of supernatant was added to 2.9 ml 50 mmol/l
potassium PBS (pH 6.0) containing 0.167 mg/ml o-dianisidine and 0.003% H2O2. Absorbance of 450 nm of
visible light (A450) was measured for 90 s.
MPO activity per gram of lung tissue was calculated as follows: MPO
activity (U/g tissue) = (dA450 × 13.5)/lung weight, where dA450 equals the rate
of change in absorbance at 450 nm between 0 and 90 s. The
coefficient 13.5 was empirically determined so that one unit of MPO
activity is the amount of enzyme that will reduce 1 mmol peroxide/min.
Statistical analysis. All results are expressed as means ± SE. Student's t-test for paired and nonpaired data and ANOVA were used to analyze the results. Regression analysis was used in the ELISA determinations. Statistical significance was set at P < 0.05.
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RESULTS |
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Portal hypertensive rats presented reduced leukocyte rolling, adhesion, and migration in mesenteric venules after LPS administration. Two weeks after the portal vein was ligated, PPVL rats showed higher portal pressure (14.8 ± 2.8 vs. 8.5 ± 1.1 mmHg, P < 0.05) and lower mean arterial pressure (90 ± 3 vs. 110 ± 4 mmHg, P < 0.05) than sham-operated rats. The weight of animals at the time of the study was similar: 367 ± 42 g in PPVL rats and 370 ± 37 g in sham-operated animals. LPS treatment (150 µg/kg) had no significant effect on blood pressure, and the significant differences between PPVL and sham-operated animals were maintained (data not shown). The number of circulating leukocytes was reduced by LPS injection (3 h) in both groups of rats [from 6.4 × 106 ± 0.7 × 106 to 4.3 × 106 ± 1.1 × 106 cells/ml in sham-operated animals (P < 0.05) and from 7.0 × 106 ± 0.9 × 106 to 3.6 × 106 ± 0.9 × 106 cells/ml in PPVL animals (P < 0.05)], and no significant differences were observed between the two groups at the same time point.
Although mesenteric venules with similar diameters were chosen for the studies in both groups, venular shear rate (
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Increased L-selectin shedding in portal hypertensive rats after LPS
administration.
To determine whether the leukocyte adhesion molecule L-selectin was
related to the defective adhesion observed in PPVL rats, the expression
of this molecule on the cell surface of neutrophils and its soluble
form were analyzed. L-selectin expression on neutrophils was similar in
both groups: 945 ± 122 mean fluorescence (MF) in PPVL vs.
932 ± 113 in sham-operated rats. However, after LPS
administration to sham-operated and PPVL rats, neutrophil L-selectin
shedding, determined as the percentage of decreased cell surface
expression, was increased in a time-dependent manner (Fig.
3). PPVL rats exhibited significantly
higher shedding of L-selectin 60, 120, and 180 min after LPS
stimulation (Fig. 3). Concomitantly to the decrease of L-selectin on
the neutrophil cell surface, soluble L-selectin levels increased in the
plasma of PPVL and sham-operated rats as measured by ELISA. However,
this increase was higher in PPVL rats (Fig.
4). Three hours after LPS administration,
soluble L-selectin levels increased 60% (from 880 ± 70 to
1,400 ± 103 U) in PPVL vs. 40% in sham-operated animals (from
650 ± 109 to 909 ± 127 U; P < 0.05, Fig.
4). These results show that L-selectin shedding was increased in PPVL
rats after endotoxin injection.
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Increased response of monocytes and neutrophils to cell activation is responsible for the altered L-selectin shedding in portal hypertensive rats. To examine whether the increase in soluble L-selectin observed in PPVL rats was due to an enhanced response of leukocyte-to-cell activation, L-selectin expression on isolated peripheral blood leukocytes from PPVL and sham-operated animals was analyzed after phorbol ester (PMA) in vitro activation.
Before activation, L-selectin surface expression was not significantly different on neutrophils, monocytes, and lymphocytes in either PPVL or sham-operated animals, as indicated by MF values (252 ± 53 vs. 278 ± 44, 191 ± 38 vs. 184 ± 37, and 189 ± 21 vs. 200 ± 57 MF, n = 6, respectively). PMA-induced L-selectin shedding from neutrophils and monocytes was significantly higher in PPVL than sham-operated rats (Fig. 5). However, no differences in lymphocyte L-selectin shedding were observed between PPVL and sham-operated rats when different doses of PMA were used (Fig. 5). Therefore, these in vitro experiments suggest that the increased shedding of L-selectin in PPVL rats was mainly due to a higher response of monocytes and neutrophils to cell activation.
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Effects of LPS injection on TNF- plasma levels.
TNF-
levels rapidly increased with a peak at 60 min, which returned
to baseline levels 180 min after administration (Fig. 7). Sixty minutes after LPS injection
(150 µg/kg), TNF-
plasma levels were higher in PPVL than in
sham-operated rats. These results are similar to those found in
previous studies.
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Neutrophil migration to the liver is not decreased in PPVL rats.
The liver is a major target of endotoxin-induced injury
(12). To test the impact of the alterations in adhesion
molecules observed in PPVL rats, we analyzed neutrophil emigration into liver sinusoids. Neutrophil emigration, measured 3 h after
endotoxin injection, was slightly higher in PPVL than sham-operated
rats, although this difference was not statistically significant (Fig. 8). Hence, endotoxin-induced leukocyte
emigration in the liver is different from the reduced adhesion and
migration of leukocytes in the mesenteric vasculature. These
alterations may reproduce endotoxin-mediated liver injury in infections
derived from enteric organisms.
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MPO levels in the lung do not change in PPVL rats compared with
sham-operated rats.
To determine whether the nonimpaired recruitment observed in the liver
in PPVL rats is comparable with LPS-induced leukocyte accumulation in
other tissues, MPO levels in lung were studied. MPO levels correlate
closely with the number of tissue neutrophil infiltration. Lung MPO
levels were similar in both groups of animals under baseline conditions
(Fig. 9). LPS administration induced a
significant increase in lung MPO activity, but no differences between
sham-operated and PPVL rats were observed.
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DISCUSSION |
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Leukocyte adhesion to vascular endothelium is critical in the development of inflammatory responses and is controlled by adhesion molecules expressed on both leukocytes and endothelial cells. In a number of tissues, leukocyte migration across endothelium requires three consecutive steps: 1) rolling, 2) firm adhesion, and 3) transendothelial migration. Rolling is mediated by selectins expressed on leukocytes (L-selectin) and activated endothelial cells (P- and E-selectin) and their mucin ligands, whereas adhesion and subsequent emigration are dependent on integrins expressed on leukocytes and immunoglobulin superfamily members, such as ICAM-1 on the endothelial cells (27, 31). Endotoxin, which acts directly on the endothelium and indirectly through the induction of cytokines by monocytes/macrophages, is a potent stimulus for the induction of several adhesion molecules. The activation and upregulation of the expression of such molecules mediate the massive emigration of leukocytes to several tissues and are the cause of the multiorgan failure observed in sepsis (17, 18).
In the present study, endotoxin caused an increase in leukocyte rolling, adhesion, and migration in the mesenteric venules of control, sham-operated rats, but this response was significantly decreased in PPVL rats. Leukocyte rolling was dramatically decreased in these rats, with numbers of rolling cells below baseline levels. Firm leukocyte adhesion and emigration were also found to be decreased in PPVL rats. Such reductions in adhesion occur despite the overexpression of the integrin CD11b and its natural ligand ICAM-1, which has been observed in these rats after endotoxin exposure (21, 22). Therefore it is likely that the reduced adhesion and subsequent emigration observed in the PPVL rats was a direct consequence of impaired rolling. These results are consistent with previous findings, which showed a defective leukocyte recruitment in the same experimental model after stimulation with PAF and LTB4 (20), thus demonstrating that impaired leukocyte recruitment occurs with other inflammatory stimuli. One major consequence of reduced leukocyte adhesion to the mesenteric venules of PPVL may be a deficient local immune response, which could facilitate the spreading of bacteria to the bloodstream. This phenomenon may explain the higher predisposition of patients with portal hypertension to develop bacterial infections from enteric organisms that could lead to septic shock.
We further studied the expression of L-selectin because it has been determined in several inflammatory models that this adhesion molecule is a major contributor to leukocyte rolling in mesenteric venules in the time frame used in our studies (1-3 h) (3, 6, 13, 16). In fact, leukocyte rolling in mesenteric venules is prevented 90 min after LPS induction by the L-selectin MAb (3, 13). The observation that the number of rolling leukocytes found in the LPS-stimulated PPVL rats was lower than that observed under baseline conditions suggests that the levels of the constitutively expressed L-selectin could be altered. The finding that the expression of cell surface L-selectin was significantly reduced in PPVL compared with sham-operated rats after endotoxin treatment supports the hypothesis that L-selectin may be responsible for the reduction of the leukocyte rolling observed in these animals. However, the possibility of the involvement of other adhesion molecules cannot be excluded. In this study, the monitoring of soluble plasma levels of L-selectin showed that the lower levels of cell surface-bound L-selectin were associated with higher levels of its soluble form in plasma. L-selectin expression is regulated by its rapid shedding from the cell surface of leukocytes on cell activation (14, 15). Recently, with the use of several protease inhibitors, it has been shown that L-selectin shedding during rolling interactions may be physiologically crucial for limiting leukocyte aggregation and accumulation at sites of inflammation (10, 34). Moreover, it has been shown that soluble L-selectin binds to its natural ligands, thus inhibiting leukocyte adhesion, which may affect the outcome of several human diseases (5, 26, 29). It is therefore conceivable that both the reduction of L-selectin expression and the increase in soluble L-selectin could contribute to the decrease of leukocyte adhesion observed in PPVL rats.
In a previous report, we showed that isolated monocytes from PPVL rats
were hypersensitive to LPS, as measured by TNF- secretion (22). Recently, we also have shown that isolated
neutrophils of PPVL rats exhibited increased L-selectin shedding and
CD11b upregulation in response to PAF and LTB4
(20). To elucidate whether the enhanced leukocyte
activation observed in endotoxin-treated PPVL rats is a general
mechanism, PMA was used in vitro as a broad cell activator causing
direct stimulation of protein kinase C. In this report we show for the
first time that monocytes and neutrophils but not lymphocytes are
hyperreactive to PMA, as measured by an overexpression of CD11b and an
increased shedding of L-selectin in PPVL rats. These data further
support the idea that the pathophysiological condition of portal
hypertension determines the priming of leukocytes to respond to
different stimuli, and this leukocyte hypersensitivity is directly
responsible for the increased shedding of L-selectin and the
hyperproduction of TNF-
.
On the other hand, it has been shown that neutrophils contribute
significantly to liver injury in experimental models of endotoxin shock
(11). Previous work has shown a major role of the adhesion molecules ICAM-1 and 2-integrins (CD11a and CD11b), but
a minimal role of selectins in the emigration of leukocytes to the
liver sinusoids (2, 9, 12, 35). The observation that
selectins are not an essential step for leukocyte migration into the
liver microvasculature and the finding that CD11b was upregulated in the leukocytes of PPVL rats led us to analyze LPS-induced migration in
liver sinusoids. Our results show that in contrast to the blunted emigration observed in the mesenteric vasculature, the number of
neutrophils migrating in sinusoids was not affected or slightly increased in PPVL rats. Moreover, neutrophil infiltration of the lung
measured by MPO activity was similar in sham-operated and PPVL rats
after LPS administration. These results are concordant with the
previous observation that mediators such as LTC4 or
endotoxin, which induced selectin-dependent rolling in numerous
vascular beds, induced selectin-independent adhesion in the sinusoids
(2, 7). However, because the number of infiltrating
leukocytes is not significantly different in PPVL compared with
sham-operated animals, the increased liver damage in response to
endotoxin may be related to the enhanced responsiveness of neutrophils
to inflammatory mediators in addition to the higher levels of TNF-
.
Our data further show that the reduction in the expression levels of L-selectin and the overexpression of CD11b in the monocytes and neutrophils may have distinct consequences depending on the vascular bed during an inflammatory response.
In conclusion, this study shows that alterations of leukocyte activation and adhesion molecule expression observed in endotoxin-stimulated PPVL rats may explain the higher susceptibility and severity of bacterial infections in patients with cirrhosis and portal hypertension.
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ACKNOWLEDGEMENTS |
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We thank Isabel Sánchez for technical assistance with these experiments
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FOOTNOTES |
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This study was supported by grants from Comisión Interministerial de Ciencia y Tecnología (SAF 96-0120, SAF 99-0007, and FIS 00-0995) and Fundació Clínic per a la Recerca Biomèdica. S. Pérez-del-Pulgar is a recipient of a grant from Formación del Personal Investigador (FP95 46731316). P. Pizcueta is a recipient of a career development award from Fondo de Investigación Sanitaria (FIS).
Address for reprint requests and other correspondence: P. Pizcueta, Fundació Clínic per a la Recerca Biomèdica, C/Villarroel 170, 08036 Barcelona, Spain (E-mail: pizcueta{at}medicina.ub.es).
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 24 May 2000; accepted in final form 24 January 2001.
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