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Departments of
*
Pharmacology and
Pathology, University of Illinois College of Medicine, and
Rush-Presbyterian-St. Luke’s Medical Center, Chicago, IL 60612
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2
integrins) with ICAM-1 enables the PMN to adhere firmly to the vascular
endothelium and thereby to migrate across the microvascular barrier
(15, 18, 19).
Recent studies have focused on the role of CD18 integrin-independent
mechanisms of PMN sequestration and emigration in the lung (15, 20, 21, 22, 23, 24, 25); however, the relative importance of these mechanisms in
mediating sepsis-induced lung microvascular injury and edema formation
in vivo remains unclear. In addition, the specific role of CD18
integrins in mediating PMN sequestration and directing PMN traffic into
lung tissue is uncertain (5, 13, 25). It is known that PMN
can engage CD18-dependent as well as CD18-independent pathways
depending on the nature of the bacterial stimulus (10, 19, 20, 22, 26, 27). Because of the complexity of PMN adhesive and
migratory events in intact lungs (17, 25, 26), there are
several questions concerning the time course of PMN sequestration and
the mechanisms of increased lung microvascular permeability and edema
formation induced by Gram-negative sepsis. Are CD18-independent
mechanisms of PMN extravasation activated in a time-dependent manner
after the onset of sepsis and are they involved in mediating the
increase in lung vascular permeability? Are these mechanisms
responsible for amplifying PMN sequestration in lung tissues? What are
the relative roles of CD18-dependent and -independent mechanisms in
mediating PMN sequestration in lung tissue and migration into the
airspace? What are the contributions of CD18-dependent and -independent
mechanisms in mediating increased microvascular permeability and edema
formation? To address these questions, we have used an in vivo mouse
model in which we determined the role of CD18 integrins and ICAM-1 in
mediating PMN sequestration and migration and increased lung
microvascular permeability and edema formation. Studies were made using
Abs against CD18 and ICAM-1 as well as in situ expression of the
specific 2 integrin binding protein neutrophil
inhibitory factor (NIF) in pulmonary microvessel endothelial cells. NIF
was used because it prevents PMN adhesion to the endothelium by
binding to CD11a and CD11b subunits in PMN (28, 29, 30). The
results show that CD18/ICAM-1-independent mechanisms capable of
markedly amplifying PMN sequestration and migration responses in lungs
are activated in a time-dependent manner after Gram-negative sepsis. We
also show that the increases in lung microvascular permeability and
lung water content are solely the result of CD18 integrin/ICAM-1
interactions even when PMN sequestration and migration responses are
markedly independent of CD18 integrins.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pathogen-free CD-1 male mice (n = 273), weighing 30–35 g and 6–12 wk old, were used in all experiments. Mice were housed in specific pathogen-free conditions at the University of Illinois animal care facility (Chicago, IL). Studies were made in accordance with institutional and National Institutes of Health guidelines, and approval was obtained from the institutional review board.
Antibodies
Purified rat IgG and mouse IgG2a were used as the control Abs
(Sigma, St. Louis, MO). The mAb 18/2.A.8 (rat anti-mouse LFA-1;
rat IgG2a) and mAb YN1/1.7.4 (rat anti-mouse ICAM-1; rat IgG2b)
were gifts from Dr. R. Rothlein (Boehringer Ingelheim, Ridgefield,
CT).
In vivo transfer of NIF
As another approach to inhibit CD18 function, we expressed NIF in mouse lungs using liposomes as we described previously (2, 31). NIF binds to CD11b and also with lower affinity to CD11a, and thereby prevents PMN adhesion to endothelial cells (28, 29, 30). NIF cDNA (50 µg/mouse) and liposomes were prepared and injected i.v. into mice to induce NIF expression in lungs (2, 31).
Morphometric analysis of lung tissue PMN sequestration
We used a computer-based stereological method to quantify the
number of PMN in lung interstitial tissue after Escherichia
coli challenge. This assessment was made in a blinded fashion
without knowledge of tissue sections. The lungs were prepared by
inflating the mouse lungs with 10% formalin and embedding in paraffin.
Tissue blocks were sectioned (5 µm thick) and mounted onto glass
slides. The H&E-stained tissue sections were visualized using a high
magnification (x100) objective with an oil immersion numerical
aperture. The computerized optical counting system consisted of a
microscope, a computer-controlled x-y-z motorized stage, a high
sensitivity video camera, a computer-assisted image capture, and a
stereological software program from MicroBrightField. (Colchester, VT).
The instrumentation was calibrated before each measurement. The middle
region (
30 mm2) of the upper lobe of the left
lung was outlined at low magnification (x1.25). At least 5% of the
outlined region was measured with a systematic random design of
counting frames as previously described (32). The total
number of PMN in the outlined region of lung was determined using the
formula: n = 
Q- x section
sampling fraction (SSF)/area sampling fraction (ASF), where
Q- is the total number of PMN counted by
optical evaluation using a random design procedure for all
measurements. The ASF is the counting frame (6400
µm2), and the SSF is the fraction of section
sampled in the region of the lung (32).
Lung tissue myeloperoxidase (MPO) activity
We compared morphometric evaluation of lung tissue PMN number with lung tissue MPO activity. Lungs were dried and homogenized in 1.0 ml 50 mM PBS (pH 6.0) with 5% hexadecyltrimethylammonium bromide and 5 mM EDTA for quantification of PMN sequestration by MPO activity as described previously by us (2, 31). The homogenates were sonicated, centrifuged at 4 x 104 g for 20 min, and frozen and thawed twice, followed by homogenization and centrifugation. The supernatant was mixed 1/30 (v/v) with assay buffer (0.2 mg/ml o-dianisidine hydrochloride and 0.0005% H2O2), and absorbance change was measured at 460 nm for 3 min. MPO activity based on dry lung weight was calculated as the change in absorbance over time.
PMN counts in bronchoalveolar lavage (BAL) fluid
The trachea was cannulated, and BAL was performed using 1 ml PBS. BAL fluid was centrifuged for 5 min at 300 rpm using a cytospin (Shandon, Pittsburgh, PA) and BAL cells were stained with HEMA3 (Fisher, Pittsburgh, PA). PMN counts were determined by counting 300 cells/slide (2, 33).
Pulmonary microvascular permeability and isogravimetric lung water determinations
Mice were anesthetized with an i.p. injection of ketamine (60–100 mg/kg), xylazine (2–2.5 mg/kg), and acepromaxine (2–2.5 mg/kg) in PBS. The trachea was cannulated with a polyethylene tube (PE 60, BD Biosciences, Peapack, NJ) for constant positive pressure ventilation (rate of 186 breaths/min). Heparin (50 U) was injected into the jugular vein as an anticoagulant. The abdominal cavity was opened to expose the diaphragm, which was ventrally punctured and cut free from the rib cage. A thoracotomy was then performed, and two halves of the rib cage were retracted to expose the heart and lungs. To make the pulmonary artery accessible for cannulation, the heart was caudally retracted with a silk suture (6-0, Ethicon, Somerville, NJ) through the apical musculature. An incision was made in the right ventricle at the base of the pulmonary artery for introducing an arterial cannula, and another incision was made in the left atrium for drainage of venous effluent. In some preparations a left atrial catheter was inserted. A polyethylene cannula (PE 60) was advanced into the pulmonary artery via the pulmonic valve and secured by means of a suture around the pulmonary artery that included the aorta. The lungs were perfused in situ using a peristaltic pump, and ventilation was continued with room air. The heart and exsanguinated lungs were rapidly excised and transferred en bloc to a perfusion apparatus, where lung preparations were suspended from a 6-cm Perspex lever arm anchored to the sensor element of a force displacement transducer (FT03, Astro-Med, West Warwick, RI). The isolated lungs were ventilated (186/min) and perfused at constant flow (2 ml/min), temperature (37°C), and venous pressure (0 cm H2O) with a modified Krebs-Henseleit solution (34) supplemented with 5 g/100 ml BSA (fraction V, 99% pure and endotoxin-free; Sigma). Pulmonary arterial pressure was monitored throughout the experiment using a pressure transducer (model P23ID, Gould Instruments, Dayton, OH). Lung wet weight was electronically nulled when the tissue was mounted, and subsequent weight changes due to gain or loss of fluid from the lung were recorded. Lung weight and arterial pressure recordings were continuously displayed on a computer video monitor with the aid of amplifiers (Astro-Med), an analog to digital converter (Scientific Solutions, Solon, OH), and commercial software for acquisition of data (Notebook Pro for Windows, Labtech, Andover, MA). All lung preparations underwent a 20-min equilibration perfusion. Lungs that were not isogravimetric at the end of the equilibration period were discarded.
The capillary filtration coefficient (Kf,c) was measured to determine pulmonary microvascular permeability to liquid as previously described (34, 35). Briefly, after the standard 20-min equilibration perfusion, the outflow pressure was rapidly elevated by 8 cm H2O for 2 min. The lung wet weight changed in a ramp-like fashion, reflecting net fluid extravasation. At the end of each experiment, lungs were dissected free of nonpulmonary tissue, and lung dry weight was determined. Kf,c (milliliters per minuter per centimeter of H2O per grams of dry weight) was calculated from the slope of the recorded weight change normalized to the pressure change and to lung dry weight.
As another approach to assess the leakiness of pulmonary microvessels, we determined the rate of pulmonary edema formation by continuously monitoring the lung wet weight changes. The weight change of lungs obtained from different groups was followed for 90 min after beginning of the perfusion (described above). As the perfusate albumin concentration was constant at the onset of perfusion, and pulmonary arterial pressure did not change during the 90-min monitoring period, the rate and magnitude of the increase in lung wet weight (i.e., attainment of a new isogravimetric state) provided an index of permeability of pulmonary microvessels.
Western blots
We determined ICAM-1 and CD18 expression in lungs by Western blotting to relate their expression to alterations in PMN sequestration and migration as determined above. Briefly, lungs were homogenized in PBS containing protease inhibitor mixture (Sigma). Protein concentration was then measured in an aliquot of the tissue homogenate. Homogenates containing equal amounts of protein were electrophoresed on 8% SDS-PAGE gels, transferred to Immobilon-P (Millipore, Bedford, MA), blocked with 5% nonfat milk, and analyzed by Western blotting using CD18 and ICAM-1 Abs (Santa Cruz Biotechnology, Santa Cruz, CA). The amount of CD18 or ICAM-1 was quantitated by scanning densitometry and normalized to the value obtained at 0 min for comparison of protein expression.
Experimental protocols
E. coli challenge. Mice were challenged i.p. with 108 live E. coli (ATCC 25992, American Type Culture Collection, Manassas, VA). This E. coli dosage was defined as sublethal, as it did not result in death within the 12-h experimental period after challenge. Control mice were injected i.p. with an equal volume of PBS. Lungs obtained at different time points after challenge (0.5, 1, 3, 6, and 12 h) were used to assess PMN sequestration (morphometric analysis and MPO activity), PMN migration into airspaces (using BAL fluid), ICAM-1 and CD18 expression, lung microvessel filtration coefficient (Kf,c), and lung wet weight gain.
CD18 and ICAM-1 mAbs. To determine the roles of CD18 and ICAM-1 in the E. coli-induced responses, we investigated the effects of mAbs against CD18 and ICAM-1. Mice were first injected i.v. with saturating concentrations of either CD18 mAb (1 mg/kg) or ICAM-1 mAb (1 mg/kg). In some cases the effects of higher concentrations of the mAbs (2 mg/kg) were also studied, and the results were compared with those obtained using 1 mg/kg. In other cases we studied the effects of combining the CD18 mAb (1 mg/kg) and ICAM-1 mAb (1 mg/kg) to address whether the effects were the same as with each mAb alone. At 30 min after injection of mAbs, mice were challenged i.p. with 108 E. coli following the procedures described above. In the control group, mice were injected i.v. with the same dosage of purified rat IgG or mouse IgG2a (1 mg/kg).
Expression of NIF. We also evaluated the changes in PMN sequestration and microvessel permeability in lungs obtained from the transgene mice expressing NIF, an alternate method to inhibit CD18 integrins (2). At 48 h after i.v. injection of the pCMVNIF construct/liposome complex (50 µg/mouse) as previously described (2, 31), when NIF expression is maximal (2, 31), mice were challenged i.p. with 108 E. coli. Lungs were obtained and studied as described above at the different time points (0.5, 1, 3, 6, and 12 h) after E. coli challenge.
Statistical analysis
Data are expressed as the mean ± SEM. Statistical analysis was performed using two-way ANOVA and Newman-Keuls test for multiple comparisons. The numbers of experiments in the different groups are given in the figure legends. A value of p < 0.05 was used as the criterion for significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lung tissue PMN sequestration as measured morphometrically
increased after E. coli challenge in a time-dependent
manner, with a 6-fold increase from basal at 1 h (Fig. 1
A). The value peaked at 3 and
6 h (7-fold increase), and then decreased toward basal levels at
12 h (3.5-fold increase; Fig. 1A). CD18 blockade
induced by either NIF expression or CD18 mAb injection interfered with
the increases in lung tissue PMN sequestration in a time-dependent
manner. PMN accumulation was partially inhibited at all time points
after E. coli challenge, except in the first 30 min (Fig. 1A). Reductions in E. coli-induced lung tissue
PMN sequestration by CD18 blockade were 50% at 1 h,
30–40% at 3 and 6 h, and 40–50% at 12 h (Fig. 1A). These time-dependent effects of CD18 blockade on PMN
sequestration were similar to the results obtained with ICAM-1 blockade
(Fig. 1A).
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B). Reductions in E.
coli-induced PMN sequestration by CD18 blockade were 75% at
1 h, 50% at 3 and 6 h, and 60% at 12 h (Fig. 1B). Moreover, the effects of ICAM-1 blockade on MPO
activity were similar to those of CD18 blockade (Fig. 1B).
Control Ab itself did not induce PMN sequestration. Transalveolar PMN migration
Migration of PMN into the alveolar space induced by i.p. E.
coli was also time dependent. However, PMN migration was delayed
compared with PMN sequestration, but, like the sequestration response,
it increased progressively with the peak responses observed at 6 and
12 h (Fig. 2). In contrast to lung
tissue PMN sequestration (Fig. 1), PMN migration remained elevated at
12 h after E. coli challenge (Fig. 2), indicating that
PMN continued to migrate into the airspace at a time when the
interstitial tissue PMN numbers had begun to significantly
decrease.
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75% of the basal value), whereas the reductions at
3 and 6 h were 50% (p < 0.05; Fig. 2). The
12 h response was reduced by 60% of the basal value (Fig. 2).
The time-dependent changes observed with ICAM-1 mAb were similar to the
CD18 blockade findings (Fig. 2).
The effects of CD18 mAb could not be ascribed to the concentration of
mAb, because similar results were obtained by doubling the mAb
concentration (Table I). Moreover,
combining the CD18 and ICAM-1 mAbs produced the same results as using
each mAb alone (Table I), suggesting that the results were secondary to
CD18/ICAM-1 interaction. The effects could not be ascribed to
nonspecific effects of the Abs, because the control Ab itself did not
induce PMN migration (Table I).
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We observed a time-dependent increase in CD18 expression, peaking
between 3 and 6 h after E. coli challenge (Fig. 3), that paralleled the times of maximum
lung tissue PMN sequestration. CD18 expression decreased at 12 h
after E. coli challenge, paralleling the decreased lung
tissue PMN sequestration at this time (Fig. 3). In contrast, ICAM-1
expression increased at 1 h and remained elevated during the 12-h
experimental period (Fig. 3).
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E. coli increased the pulmonary microvessel
Kf,c (Fig. 4) as well as the isogravimetric lung wet
weight (Fig. 5). The increase in
Kf,c was time dependent; that is,
Kf,c increased by 150 and 280% of
baseline values at 1 and 6 h, respectively, after E.
coli challenge (p < 0.05;
n = 4 in each group). The increase in
Kf,c persisted during the 12-h
E. coli exposure period (Fig. 4).
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), consistent with the
time-dependent leakiness of pulmonary microvessels (Fig. 4). However,
lungs challenged with E. coli for 12 h gained less
water (Fig. 5), indicative of a return in vascular permeability toward
basal levels. Increases in Kf,c and
isogravimetric lung wet weight were prevented by CD18 or ICAM-1
blockade at all times after E. coli challenge (Figs. 4 and 5); that is, even when PMN sequestration and migration responses were
significantly CD18 integrin and ICAM-1 independent. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PMN adhesion and transendothelial migration responses in cell culture studies and experimental animal models of Gram-negative sepsis have been shown to be mediated through the CD11/CD18 complex and ICAM-1, its endothelial plasma membrane counter-receptor (4, 15, 18, 36). However, recent evidence indicates that PMN adhesion to pulmonary microvascular endothelial cells and migration into lung tissue can also occur by CD11/CD18-independent adhesion pathways (10, 15, 20, 21, 24, 25, 37). Anti-CD18 Abs blocked sepsis-induced PMN emigration by 60–80% (10, 15, 38), suggesting an important component of PMN migration involves CD18-independent pathways. On analyzing the time course of the responses in the present study, we showed that CD18-dependent and -independent mechanisms coexist but that their contributions varies in a time-dependent manner. The results indicated that there was time-dependent engagement of the CD18-independent PMN sequestration and migration, in that the peak responses observed at 3 and 6 h after E. coli were only 40% CD18 integrin dependent. Thus, the results support the concept that a time-dependent engagement of CD18-independent mechanisms is important in amplifying the lung tissue accumulation and airspace migration of PMN in response to E. coli challenge.
We showed that sequestration of PMN in lung tissue was coupled to their migration into the airspace; thus, the activation of CD18-independent PMN adhesive mechanisms is capable of directing PMN traffic across the capillary-alveolar barriers. Lung tissue PMN sequestration decreased at 12 h of E. coli exposure, suggesting that mechanisms capable of inactivating PMN accumulation are also engaged in a time-dependent manner. It is unlikely that a decrease in the expression of ICAM-1 is responsible for this effect, because ICAM-1 protein expression remained elevated even at 12 h after E. coli challenge. The possibility exists that antiadhesive mechanisms are activated in vivo capable of decreasing lung tissue PMN accumulation in the face of continued E. coli exposure and ICAM-1 expression. Thus, mechanisms responsible for directing PMN traffic into the airspace remained active even up to 12 h after E. coli challenge, whereas lung interstitial tissue PMN accumulation at this time had been effectively inhibited.
The present observations indicate that CD18 integrin-independent pathways engaged in a time-dependent and reversible manner are critical in amplifying PMN sequestration and migration in lungs during Gram-negative sepsis. The basis of the engagement of CD18-independent mechanisms is not clear. In an in vitro system in which CD18-indepenent pathways of transendothelial PMN migration were identified, IL-8 was shown to promote the CD18-independent emigration of PMN (8, 24, 26, 39). In another study we showed that the E. coli induced the release of macrophage inflammatory protein-2 (our unpublished observation), the murine homologue of IL-8, in lung tissue, which could account for the observed CD18-independent tissue PMN sequestration and migration responses. A recent study has shown that very late Ag-4 (CD49d/CD29) and very late Ag-5 (CD49e/CD29) may also be involved in mediating CD18-independent PMN migration in the rat lung after endotoxin-induced lung inflammation (25).
Although CD18 integrin-independent mechanisms can amplify PMN sequestration and migration in lung tissue, their contribution in mediating lung microvascular injury and edema formation in vivo is unclear. To address the roles of CD18 integrins in mediating microvessel injury and tissue edema, we used two independent in vivo approaches to block CD18 function: injection of mAbs directed against CD18 and ICAM-1 and in situ expression of NIF, a specific CD11a and CD11b peptide antagonist, in lungs (2, 28, 29, 30, 31). We showed that CD18 integrin blockade by either means prevented the increase in pulmonary capillary filtration coefficient (a measure of microvessel endothelial permeability) and edema formation at all times from 1–12 h after E. coli exposure. The protection was evident even when CD18 blockade inhibited PMN accumulation by only 40%. These results indicate the importance of PMN CD18 in the mechanism of increased lung microvessel permeability and edema formation induced by Gram-negative sepsis and support the value of blockade of CD18 activation in preventing acute lung injury.
The basis by which the E. coli-induced increase in pulmonary microvessel permeability can be fully ascribed to CD18 integrins is not clear. One possible explanation is that activation of the CD18-independent pathways is a delayed event, whereas the initial PMN sequestration depends more on CD18 integrins. As onset of the increase in endothelial permeability following activation of PMN adherent to endothelial cells is rapid (40, 41), it is likely that the initial CD18 dependence of PMN sequestration would be a critical determinant of increased microvessel permeability. Another possibility is that the initial activation of PMN CD18 integrins results in the respiratory burst and release of oxidants involved in mediating lung microvascular injury (6, 7). Thus, inhibition of CD18 function, and thereby of oxidant production, would be protective even though the subsequent PMN sequestration and migration responses rely to a greater extent on CD18-independent mechanisms.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Asrar B. Malik, Department of Pharmacology, University of Illinois College of Medicine, 835 South Wolcott Avenue (M/C868), Chicago, IL 60612. E-mail address: abmalik{at}uic.edu
3 Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; NIF, neutrophil inhibitory factor; BAL, bronchoalveolar lavage; MPO, myeloperoxidase; Kf,c, capillary filtration coefficient.
Received for publication December 28, 2000. Accepted for publication June 29, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 integrin-dependent mechanism. J. Clin. Invest. 101:2427.[Medline]
41 (very late antigen (VLA)-4, CD49d/CD29) and 51 (VLA-5:CD49.e/CD29) integrins mediate 2 (CD11/CD18) integrin-independent neutrophil recruitment to endotoxin-induced lung inflammation. J. Immunol. 166:4644.2 integrins. Mol. Pharmacol. 56:926.2-integrin blockade driven by E-selectin promoter prevents neutrophil sequestration and lung injury in mice. Circ. Res. 87:254.2-integrin CD18 in the local accumulation of neutrophils in pulmonary and cutaneous inflammation in the rabbit. Am. J. Respir. Cell Mol. Biol. 10:391.[Abstract]
2 integrins (CD11/CD18) in the regulation of cytokine gene expression of polymorphonuclear neutrophils during the inflammatory response. FASEB J. 13:1855.This article has been cited by other articles:
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, compared with no E. coli (basal
value); *, compared with E coli alone (no
pretreatment); and 
