Neutrophil adhesion is impaired in the mesentery but not in the liver sinusoids of portal hypertensive rats

Sofía Pérez-del-Pulgar1, Pilar Pizcueta1, Pablo Engel2, Jaume Bosch1, and Joan Rodés1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha ) 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-alpha and IL-6 (1). A positive correlation between the levels of TNF-alpha 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-alpha in response to endotoxin (4). We have shown that portal hypertensive rats have enhanced TNF-alpha plasma levels after LPS injection, together with a significant upregulation of CD11b/CD18 expression on monocytes (22). In addition, elevated levels of TNF-alpha 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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
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 (gamma ) was calculated from the Newtonian definition: gamma  = 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-alpha levels. TNF-alpha levels were determined using a sensitive ELISA kit (Genzyme) specific for rat TNF-alpha , 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-alpha 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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (gamma ) and VRBC were decreased in PPVL animals (Table 1). LPS administration induced a significant decrease in shear rate in the two groups (PPVL 378 ± 53 s-1 and sham-operated 481 ± 38 s-1, P < 0.05).

                              
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Table 1.   Hemodynamic parameters

Intravital microscopy studies on mesenteric venules showed that leukocyte rolling flux was similar in PPVL rats and sham-operated animals in baseline conditions. LPS administration to sham-operated rats induced a moderate increase in leukocyte rolling after 1 h, followed by a marked increase after 3 h (Fig. 1A). In contrast, leukocyte rolling in PPVL rats decreased below baseline levels after endotoxin stimulation (42 and 51% decrease vs. sham-operated rats 1 and 3 h after LPS administration, respectively). Hence, a dramatic difference between the number of rolling leukocytes was observed between PPVL and sham-operated rats. Endotoxin induced a decrease in the leukocyte rolling velocity that was similar in both groups of animals, indicating that the diminished leukocyte rolling in PPVL rats was not related to a difference in the leukocyte rolling velocity after LPS administration (Fig. 1B).


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Fig. 1.   Leukocyte rolling flux (A) and velocity of white blood cells (VWBC; B) in mesenteric venules of sham-operated and partial portal vein ligated (PPVL) rats. Measurements were made in baseline conditions and 60 and 180 min after lipopolysaccharide (LPS) injection. Results are expressed as means ± SE. *P < 0.05 vs. sham-operated rats. #P < 0.05 vs. basal.

Sham-operated and PPVL animals exhibited a marked increase in leukocyte adhesion and migration after LPS administration (Fig. 2). However, this increase was significantly lower in PPVL than in sham-operated animals. The number of migrated cells also remained significantly lower in PPVL than sham-operated animals (Fig. 2B). These results indicated that the blunted recruitment of leukocytes in mesenteric venules of PPVL rats was probably due to an impaired leukocyte rolling response, which was most likely related to an altered expression of adhesion molecules.


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Fig. 2.   Number of adherent (A) and migrated (B) leukocytes in mesenteric venules of sham-operated and PPVL rats. Measurements were made in baseline conditions and 60 and 180 min after LPS injection. Results are expressed as means ± SE. *P < 0.05 vs. sham-operated rats. #P < 0.05 vs. basal.

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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Fig. 3.   Percentage of L-selectin shedding from the cell surface of circulating blood neutrophils of PPVL and sham-operated rats 0, 30, 60, 120, and 180 min after LPS administration. Results are expressed as means ± SE. *P < 0.05 vs. sham-operated rats.



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Fig. 4.   Levels of plasma soluble L-selectin in PPVL and sham-operated animals at different times after LPS injection (150 µg/kg). Values are expressed as means ± SE. *P < 0.05 vs. sham-operated rats.

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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Fig. 5.   Percentage of L-selectin shedding from the cell surface of neutrophils (A), monocytes (B), and lymphocytes (C) of PPVL and sham-operated rats after phorbol 12-myristate 13-acetate (PMA) stimulation. Results are expressed as means ± SE. *P < 0.05 vs. sham-operated rats.

To test whether the expression of other adhesion molecules on the neutrophils and monocytes of PPVL rats was altered after cell activation, we next studied CD11b expression. As observed for L-selectin, flow cytometric analysis showed that CD11b expression on neutrophils and monocytes was similar in PPVL and sham-operated animals before stimulation MF (77 ± 4 vs. 72 ± 7 and 78 ± 4 vs. 71 ± 5, respectively; Fig. 6). PMA stimulation caused a dose-dependent increase in CD11b expression on the leukocytes from both groups of rats (Fig. 6). However, after PMA activation, the expression levels of CD11b on monocytes and neutrophils from PPVL rats were significantly higher than those from sham-operated rats (Fig. 6). These data show that the increased L-selectin shedding observed in PPVL rats is concomitant to an overexpression of CD11b. Our results also show that leukocytes from PPVL rats are not only primed to respond to endotoxin but also to other activators such as PMA.


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Fig. 6.   Percentage of increase of CD11b expression on the cell surface of neutrophils (A) and monocytes (B) of PPVL and sham-operated rats after PMA stimulation. Results are expressed as means ± SE. *P < 0.05 vs. sham-operated rats.

Effects of LPS injection on TNF-alpha plasma levels. TNF-alpha 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-alpha 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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Fig. 7.   Levels of plasma tumor necrosis factor (TNF)-alpha in PPVL and sham-operated rats at 30, 60, and 180 min after LPS infection in vivo (150 mg/kg). Results are expressed as means ± SE. P < 0.05, TNF-alpha values at 30 and 60 vs. baseline in both groups.

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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Fig. 8.   Evaluation of sinusoidal neutrophil (PMN) sequestration in PPVL and sham-operated rats 3 h after injection of LPS or vehicle (PBS). Five rats were used for each group. Five microscopic fields at the magnification of ×400 were randomly chosen from each liver, and all of the sinusoids included in the field were analyzed. #P < 0.05 vs. vehicle.

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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Fig. 9.   Myeloperoxidase (MPO) levels in PPVL and sham-operated rats 3 h after injection of LPS or vehicle (PBS). Five rats were used for each group. Results are expressed as means ± SE. *P < 0.05 vs. sham-operated rats


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha 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-alpha .

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 beta 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-alpha .

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.


    ACKNOWLEDGEMENTS

We thank Isabel Sánchez for technical assistance with these experiments


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastrointest Liver Physiol 280(6):G1351-G1359
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