Departments of 1 Pharmacology and 2 Anesthesiology, College of Medicine, The University of Illinois, Chicago, Illinois 60612
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ABSTRACT |
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We determined the time-dependent effects of conditional expression of neutrophil inhibitory factor (NIF), a specific 41-kDa CD18 integrin antagonist, on the time course of NIF expression and lung PMN (polymorphonuclear leukocyte) infiltration and vascular injury in a model of Escherichia coli-induced sepsis in mice. Studies were made in mice transduced with the E-selectin (ES) promoter-NIF construct (using liposomes) in which the NIF cDNA was driven by the inflammation- and endothelial cell-specific ES promoter. We observed time-dependent expression of NIF in pulmonary vascular endothelium that paralleled the ES expression. Expression of both was evident at 1 h after E. coli challenge, peaked at 3-6 h, and returned to basal level within 48 h. We observed that increases in PMN uptake and transalveolar PMN migration induced by E. coli challenge were reversed in a time-dependent manner following NIF expression in mice. NIF expression also prevented the progression of lung vascular injury and edema formation following E. coli challenge. Thus the conditional expression of NIF using the ES promoter can reverse, in a time-dependent manner, lung PMN infiltration and vascular injury induced by gram-negative sepsis. The results support the model that initial engagement of CD18 integrins enables the further recruitment of additional PMN into lung tissues such that PMN continue to sequester and migrate after E. coli challenge.
E-selectin promoter; gram-negative sepsis; CD18 blockade; polymorphonuclear leukocyte migration and uptake; lung vascular permeability
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INTRODUCTION |
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LEUKOCYTE TRAFFICKING IN
PULMONARY tissue and air spaces is critical in the host defense
response; however, migration and activation of polymorphonuclear
leukocytes (PMN) into lungs also contribute to the mechanism of
inflammatory tissue injury (10, 24, 25). It is well
accepted that PMN migration from the blood vessels to site of infection
occurs by a multistep process dictated by sequential activation of
adhesive proteins and their counterreceptors on PMN and endothelial
cells (3, 22, 12). Initiation of PMN migration begins with
"capture" of PMN by endothelial cells, and this is followed by PMN
"rolling" along the vessel wall mediated by adhesive proteins of
the selectin family (1, 2, 23). Rolling PMN become firmly
adherent to endothelial cells following activation of intercellular
adhesion molecule-1 (ICAM-1) (4, 9, 11). The cells are
thus positioned for migration into tissue parenchyma and then into the
alveolar space. E-selectin (ES) as well as L-selectin and
P-selectin contribute to the capture and rolling of PMN, whereas
2-integrins and ICAM-1 mediate firm adhesion of PMN to
endothelial cells (16, 21, 26).
Neutrophil inhibitory factor (NIF), a 41-kDa glycoprotein isolated
from the canine hookworm (Ancylostoma caninum), was
identified as a potent inhibitor of PMN adhesion to endothelial cells
and adhesion-dependent release of hydrogen peroxide (17).
These effects of NIF result from its specific binding to the I-domain of CD11/CD18 2-integrins (15, 17, 18, 28).
NIF binds to CD11b with high affinity and CD11a with lower affinity
(15) and prevents PMN adhesion to endothelial cells to a
degree equivalent to anti-CD18 monoclonal antibodies (15).
We have shown that conditional expression of NIF using the E-selectin
promoter (pES) is a useful strategy in preventing PMN adhesion to
activated endothelial cells and resultant lung microvascular injury
(27). However, previous studies have not examined the time
course of the response, i.e., the temporal relationship between
expression of ES and NIF and the relationship between NIF expression
and reversal of PMN infiltration and lung microvessel injury. In the
present study, we used ES promoter to induce NIF expression since ES
transcription is activated specifically in endothelial cells in
response to inflammatory stimuli such as gram-negative septicemia
(19).
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METHODS |
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Mice. Pathogen-free CD1 mice (males; 30-35 g body wt; Harlan, Indianapolis, IN) were used for the in vivo experiments. These mice have been extensively used in models of endotoxemia. Moreover, we established the liposome-based method of gene delivery in these mice. Therefore, we have used these mice in the present study to express NIF as well as to examine Escherichia coli responses. Mice were housed in pathogen-free conditions at the University of Illinois Animal Care Facility where they were treated in accordance with institutional and National Institutes of Health guidelines. Before injection of transgene/liposome complex, mice were anesthetized with intramuscular injection of ketamine (60 mg/kg) and xylazine (2 mg/kg) in PBS.
DNA/liposome preparation and in vivo gene transfer. Dimethyldioctadecylammonium bromide (DDAB; Sigma Chemical, St. Louis, MO) and cholesterol (Calbiochem, La Jolla, CA) in chloroform were used to prepare the liposomes as described (6, 14, 29). Briefly, the mixture consisting of DDAB and cholesterol (1:1 molar ratio) was dried using a Rotavapor R-124 (Brinkmann, Westbury, NY) and dissolved in 5% glucose. The lipid molecules were sonicated for 20 min. The construct pES-NIF in which NIF cDNA (a kind gift of Corvas, La Jolla, CA) is driven by the ES promoter was made as described (27). The pES-NIF construct (50 µg/mouse) and liposomes were combined at a ratio of 1 µg of DNA to 8 nmol of liposomes and injected intravenously into mice.
Challenge of mice with E. coli. Mice received 1 × 108 live E. coli (American Type Culture Collection 25992) in PBS (pH 7.4) via intraperitoneal injection. The E. coli dosage was chosen because it did not result in death of mice within the 48-h experimental period. Control mice were injected intraperitoneally with an equal volume of PBS. Mortality studies were carried out in another group of mice challenged with 1 × 109 live E. coli injected intraperitoneally.
Western blot analysis. Mouse lungs were homogenized in PBS (pH 7.4) containing protease inhibitor cocktail (66 µl/10 ml PBS, Sigma) using a tissue grinder at a ratio of 1 g of lung to 5 ml of PBS. The homogenates were centrifuged at 1,000 rpm for 10 min at 4°C. Supernatants were collected, and the protein concentration of each sample was measured with Non-Interfering Protein Assay (Geno Technology, St. Louis, MO). An equal amount of protein from each sample (~50 µg) was resolved in 8% SDS-PAGE gel, and Western blot analysis was performed to determine the expression of ES and NIF as described (20). A polyclonal antibody against ES was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A polyclonal antibody against NIF was made using a peptide containing the COOH-terminal sequence of NIF (IPDDGVCFIGSKADYDSKEFYRFREL). Densitometry of bands was performed using Scion Image software (Scion, Frederick, MD).
Immunocytochemistry. Lung sections (4 µm) of mice were prepared as previously described (29) and stained with rabbit anti-NIF antiserum using the ImmunoCruz Staining System (Santa Cruz Biotechnology).
Lung PMN sequestration. Mouse lungs were homogenized in 5% hexadecyltrimethylammonium bromide (HTAB) in 50 mM phosphate buffer (pH 6; 5.0 ml of HTAB/g tissue) for quantification of PMN uptake by myeloperoxidase (MPO) activity (13). The homogenates were centrifuged at 40,000 rpm for 30 min. The pellets were subjected to three cycles of freeze-thaw; each cycle was followed by homogenization and centrifugation. The three supernatants were collected and mixed; 0.1 ml of the pooled sample was mixed 1:30 (vol/vol) with assay buffer (0.167 mg/ml of O-dianisidine hydrochloride and 0.0005% H2O2), and the absorbance change was measured at 460 nm for 3 min. MPO activity was calculated as the change in absorbance over time.
PMN counts in bronchoalveolar lavage fluid. Bronchoalveolar lavage (BAL) was performed by cannulating the trachea with a blunt-ended 21-gauge needle, instilling 0.6 ml of sterile PBS containing 1 mM EDTA, and collecting the fluid by gentle aspiration. The total fluid (0.5 ml) was centrifuged for 5 min at 300 rpm using a cytospin3 (Shandon, Pittsburgh, PA), and BAL cells were stained with HEMA3 (Biochemical Sciences, Swedesboro, NJ). The total number of cells in BAL (which consisted mostly of macrophages and PMN) per slide were counted. Each slide was divided into 20 fields, and the total number of cells was calculated by multiplying the average number of cells in one field by 20. The number of PMN as a percentage of the total cells was calculated.
Pulmonary microvascular permeability and isogravimetric lung
water determinations.
Capillary filtration coefficient (Kfc) was
measured to determine pulmonary microvascular permeability to liquid as
described (7). Briefly, after the standard 20-min
equilibration perfusion, the outflow pressure was rapidly elevated by
10 cmH2O for 2 min. The lung wet weight changed in a
ramplike fashion, reflecting net fluid extravasation. At the end of
each experiment, lungs were dissected free of nonpulmonary tissue, and
lung dry weight was determined. Kfc
(ml · min1 · cmH2O · g
dry wt
1) was calculated from the slope of the
recorded weight change normalized to the pressure change and to lung
dry weight.
Survival studies in mice challenged with E. coli. At 48 h after intravenous injection of pES-NIF/liposome complex, mice were challenged with a dosage of 109 E. coli per mouse intraperitoneally for mortality analysis. Mice not receiving injection of NIF construct were used as controls. A 50% survival rate after E. coli challenge was determined in all groups.
Statistical analysis. Data are expressed as means ± SE. Comparisons between experimental groups were made by ANOVA with a significance value set at P < 0.05.
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RESULTS |
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Time course of ES and NIF expression in mouse lungs induced with E. coli challenge.
In the group of mice challenged with intraperitoneal E. coli
(108 live E. coli), Western blot analysis showed
that ES expression was induced in a time-dependent manner after
challenge with E. coli. ES expression was seen as early as
1 h, peaked at 6 h, and decreased at 48 h (Fig.
1A). The time course of ES
promoter-driven NIF expression was similar to ES (Fig. 1,
A and B). Western blot and immunohistochemical
analyses of lungs from the pES-NIF-transduced mice showed that NIF
expression peaked between 3 and 6 h and returned to basal levels
by 24 h after E. coli challenge (Fig. 1, A
and B).
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Time-dependent effects of conditional NIF expression on PMN uptake
and migration in mouse lung.
Lung MPO activity in control mice increased within 1 h after
E. coli challenge, peaked at 6 h, and returned to
baseline at 24 h (Fig. 2). MPO
activity of lungs conditionally expressing NIF after the E. coli challenge was reduced in a time-dependent manner by
65-90% of the control values (Fig. 2). This temporal reduction in
MPO activity paralleled the time course of NIF expression (Fig. 1).
Analysis of BAL fluid showed that the percentage of PMN in BAL fluid
increased after challenge of E. coli; the response peaked at
12 h (11.2% of total recoverable cells were PMN) and returned
toward baseline by 48 h (Fig.
3A). In the group of mice in
which lungs were transduced with pES-NIF, the increase in PMN in BAL
fluid was significantly reduced in a time-dependent manner (by
75-100% of control values; Fig. 3A), paralleling the
NIF expression (Fig. 1). The total number of cells in BAL fluid (which
consisted primarily of macrophages and PMN) showed the same
time-dependent changes (Fig. 3B).
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Time-dependent NIF expression prevents increased lung vascular
permeability and edema formation induced with intraperitoneal E. coli.
We quantified microvessel liquid permeability (as
Kfc) and changes in isogravimetric lung water
content to address the time-dependent effects of conditional NIF
expression on PMN-mediated lung microvessel injury. E. coli
challenge resulted in time-dependent increases in extravascular lung
water content (Fig. 4A) and
lung microvessel permeability (Fig. 4B). The increases in
lung water content and vascular permeability peaked 6 h after
E. coli. Time-dependent expression of NIF reversed the
progressive permeability increase and edema induced by E. coli in control mice (Fig. 4).
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Conditional NIF expression reduces mortality induced with lethal
dosage of E. coli.
We determined whether expression of NIF reduced mortality of mice
challenged with E. coli. Because the sublethal dosage of E. coli did not produce death within 48 h of E. coli challenge, we used the 10-fold higher lethal dosage of
E. coli in these mortality experiments. At 48 h after
expression of pES-NIF, mice were challenged with 109
E. coli per mouse via intraperitoneal injection. Mice not
receiving injection of NIF construct were used as controls. In the
group of mice challenged with E. coli, 50% survived in the
control group at 6 h, whereas 50% of the pES-NIF-expressing mice
were alive at 8 h (Fig. 5). All
control mice were dead at 9 h after E. coli challenge,
whereas 40% of pES-NIF-expressing mice were alive at this time (Fig.
5). Together, the data demonstrate that inducible expression of NIF not
only prevents progression of lung vascular injury and edema formation
but also reduces the mortality rate after E. coli challenge.
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DISCUSSION |
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We reasoned that it would be possible to, in a time-dependent
manner, arrest the progression of PMN uptake and increased lung microvessel permeability using the endothelial cell-selective ES
promoter to express the specific CD18 antagonist NIF. Because ES is
expressed in endothelial cells by inflammatory stimuli such as
gram-negative sepsis (5), this would be a potential means of conditionally and temporally preventing lung microvessel injury in
an inflammation-specific manner. The results indicate that NIF
expression had a similar time course as expression of ES and that the
expression of NIF was silenced as ES expression decreased to basal
levels. In the present study, we show the reversibility of lung tissue
PMN uptake and transalveolar PMN migration and lung microvessel injury
after E. coli-induced gram-negative septicemia. In previous
studies, we showed that NIF can bind to CD11b with high affinity and to
CD11a with low affinity (15) and that it thereby prevents
PMN adhesion to endothelial cells to a degree equivalent to anti-CD18
monoclonal antibodies (15). These effects of NIF result
from its specific binding to the I-domain of CD11/CD18 2-integrins (15, 17, 18, 28).
To study the time-dependent effects of the conditional NIF expression, we used the intraperitoneal E. coli challenge mouse model (108 live E. coli). E. coli challenge resulted in marked lung tissue PMN sequestration but with relatively few PMN migrating into the air space (at peak response, ~11% of cells in BAL fluid were PMN). These responses were time dependent in that PMN accumulated in lung interstitial tissue as early as 1 h after E. coli challenge, reached a peak value at 6 h, and returned to basal levels between 12 and 24 h. PMN migration into lung air space was, however, delayed and remained elevated for up to 12 h. The decrease in lung PMN uptake at 12 h suggests the existence of "antiadhesive" mechanisms, which are activated in vivo and capable of decreasing lung PMN uptake in the face of continued E. coli exposure. Thus the mechanisms responsible for PMN migration into the air space remained active even up to 12 h after E. coli challenge, whereas lung tissue PMN uptake at this time had been significantly inhibited.
We have shown in other studies that intratracheal E. coli challenge results in a peak response at which ~90% of cells in the air space are PMN (unpublished observations). Thus the route of E. coli challenge is an important determinant of transalveolar PMN migration. Nevertheless, the magnitude of PMN sequestration and migration after intraperitoneal E. coli challenge was sufficient to induce an approximate twofold increase in the Kfc as early as 1 h post E. coli challenge. This finding suggests that the initial PMN uptake after the onset of septicemia is capable of inducing severe lung microvessel injury. Capillary filtration increased further at 6 h post-E. coli, indicating the progression of lung microvessel injury as PMN continued to accumulate in lungs in a time-dependent manner. Our finding that the pulmonary capillary coefficient returned to the basal value at 24 h after 108 E. coli challenge indicates that the endothelial barrier in this sublethal model is capable of reestablishing its barrier property.
NIF expression induced by E. coli prevented, in a time-dependent manner, PMN uptake by 65-90% and transalveolar PMN migration by 75-100% after the E. coli challenge. This finding demonstrates that CD18 blockade, as induced by the conditional release of NIF, can arrest the PMN uptake and transalveolar PMN migration responses. The results can be explained by the finding that NIF is released in the pulmonary microcirculation and binds to CD18 integrins on leukocytes (29) and thus prevents the progressive infiltration of PMN in lung tissue. The finding that NIF prevents the progression of PMN uptake and migration within the first 3 h of its expression suggests that CD18-dependent mechanisms are critical in mediating initial PMN uptake and migration responses in lungs. The results support the model that initial engagement of CD18 integrins enables the further recruitment of additional PMN into lung tissues such that PMN continue to sequester and migrate during the 6- to 12-h period following E. coli challenge.
Lung wet weight and Kfc were monitored to determine the time course of edema formation and increased pulmonary microvascular permeability, respectively. Previous studies have shown that these responses are dependent on CD18 integrins since CD18 antibodies prevented lung microvessel injury in this model (8). In the present study, we show in mice challenged with intraperitoneal E. coli that lung wet weight and Kfc increased progressively and peaked at 6 h, reflecting the time-dependent nature of lung microvessel injury. This finding indicates lung microvessel injury parallels the infiltration of PMN in lungs. Expression of NIF using the pES-NIF construct prevented the progressive increase in lung vascular permeability and edema formation, indicating that these responses were CD18 integrin dependent. Thus CD18 blockade, induced in a conditional manner, may enable the repair of lung microvascular injury by preventing the progressive infiltration of PMN.
Although our previous studies have shown a protective effect of NIF expression in mice (27, 29), the present studies extend these observations by demonstrating the time-dependent nature of the response in which conditional NIF expression was coupled to reversal of lung tissue PMN uptake and transalveolar migration as well as lung microvessel injury. The conditional expression of NIF also significantly reduced mortality in mice challenged with a 10-fold higher dosage of E. coli than the sublethal dosage used for the functional studies.
In summary, findings based on the conditional time-dependent expression of the selective CD18 integrin antagonist NIF suggest that engagement of PMN CD18 is required for the increases in PMN sequestration, transalveolar PMN migration, and increased lung microvessel permeability induced by gram-negative septicemia. Gene transfer based on gram-negative septicemia-induced expression of NIF may be useful in reversing lung PMN infiltration and lung vascular injury and edema formation. The advantage of this approach is that it is not only conditional (i.e., requiring the inflammatory stimulus for NIF induction) but also endothelial cell specific (it relies on ES promoter) and transient (it parallels ES expression).
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-64573, HL-46350, and HL-45638.
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FOOTNOTES |
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Address for reprint requests and other correspondence: A. Rahman, Dept. of Pharmacology, College of Medicine, The Univ. of Illinois, 835 S. Wolcott Ave., Chicago, IL 60612-7343 (E-mail: ARahman{at}uic.edu).
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.
10.1152/ajplung.00298.2001
Received 2 August 2001; accepted in final form 13 November 2001.
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