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Departments of 1 Internal
Medicine and 2 Biochemistry, This study aimed
to examine the behavior of stimulated leukocytes in the pulmonary
microcirculation. The leukocyte-endothelium interaction was visualized
under physiological shear rates in perfused rat lungs using high-speed
confocal laser video microscopy. Leukocytes labeled with
carboxyfluorescein were stimulated with cytokine-induced neutrophil
chemoattractant (CINC/gro), which caused L-selectin shedding and
inverse upregulation of CD18. Neither unstimulated nor stimulated
leukocytes exhibited rolling in either pulmonary arterioles or venules,
whereas both were sequestered in capillaries. Approximately 50% of
stimulated leukocytes showed a transient cessation of movement in
pulmonary capillaries. The CINC/gro stimulation, which inhibited
leukocyte rolling and adhesion to mesenteric venules, reduced leukocyte
velocity and increased leukocytes in pulmonary capillaries.
Pretreatment with monoclonal antibodies against intercellular adhesion
molecule-1 (ICAM-1) or CD18 attenuated these changes. Confocal
microfluorography revealed constitutive expression of ICAM-1 not only
in venules but also abundantly in capillary networks. These results
suggest that selectin-independent, CD18-ICAM-1-dependent capillary
sequestration is one of the major mechanisms by which activated
leukocytes accumulate in the lungs.
pulmonary microcirculation; mesenteric microcirculation; L-selectin; leukocyte adhesion; leukocyte plugging
TISSUE LEUKOCYTE accumulation is considered to occur
via sequential multistep mechanisms, including rolling, adhesion, and migration, which involve different classes of adhesion molecules (6,
22). In a variety of disease conditions such as acute inflammation and
ischemia-reperfusion, neutrophil adhesion and transendothelial
migration have been shown to be mediated through CD11/CD18 and its
endothelial ligand intercellular adhesion molecule-1 (ICAM-1) in vivo
(13, 29) and in vitro (20).
In pulmonary microvessels, however, whether these adhesion molecules
are required for leukocyte accumulation is still controversial (4, 10).
Worthen et al. (35) reported that stimulated neutrophils remain in
pulmonary capillaries as a result of decreased cellular deformability.
Vedder et al. (28) demonstrated that blockade of the CD11/CD18
glycoprotein adherence complex by the monoclonal antibody (MAb) 60.3 inhibited neutrophil accumulation in the gut but did not inhibit the
pulmonary sequestration of neutrophils in ischemia-reperfusion injury.
In addition, recent data obtained from studies utilizing P-selectin and
ICAM-1 double mutant mice showed that peritoneal neutrophil emigration
and edema formation induced by Streptococcus
pneumoniae were attenuated in mutant mice, whereas
pulmonary neutrophil emigration and edema formation occurred as
observed in the wild-type mice (4). These results suggest little, if
any, involvement of these adhesion molecules in the mechanism
underlying pulmonary leukocyte accumulation and in the subsequent lung
injury. On the other hand, several previous studies have shown that
anti-CD18 MAb attenuated lung injuries induced by tumor necrosis factor
(17), gram-negative sepsis (31), and zymosan-activated plasma (8).
Although the discrepancy concerning the contribution of the CD18-ICAM-1
interaction to neutrophil accumulation in the lung appears to reflect
the varied experimental protocols and choices of animal species used,
the crucial factors leading to this controversy are possible
differences in wall shear rates and circulating leukocyte numbers among
the experimental models as well as unique features of the interaction
between activated leukocytes and endothelium in the pulmonary
microcirculation. We hypothesized that sequential multistep
leukocyte-endothelium interactions are not applicable to lung
microvessels. Therefore, the aim of the present study was to examine
the behavior of circulating leukocytes and their adhesion mechanisms in
the rat pulmonary microcirculation perfused ex vivo under controlled
flow conditions.
Reagents used.
Rat cytokine-induced neutrophil chemoattractant (CINC/gro) is a peptide
possessing biological activities analogous to those of the human
interleukin-8 (IL-8) family (32) and was a generous gift from Dr. K. Watanabe (Institute of Cytosignal Research, Tokyo, Japan). MAbs against
CD18 (WT-3), against ICAM-1 (1A29), and against L-selectin (HRL-4) were
generously provided by Dr. M. Miyasaka (Dept. of Bioregulation, Osaka
University Medical School, Biomedical Research Center, Osaka, Japan).
Fluorescein isothiocyanate (FITC), FITC-dextran (mol wt 145,000),
FITC-labeled anti-mouse immunoglobulin G (IgG) antibody, and mouse IgG
were purchased from Sigma (St. Louis, MO), and carboxyfluorescein
diacetate succinimidyl ester (CFSE) was from Molecular Probes (Eugene,
OR). MAb (2H5) against sialyl Lewis X-like carbohydrates (SLeX) was
provided by Dr. T. Tamatani (Pharmaceutical Basic Research
Laboratories, Japan Tobacco, Yokohama, Japan) (27).
Animal preparation.
Specific pathogen-free male Sprague-Dawley rats (Sankyo Laboratory
Service, Tokyo, Japan), 8 wk of age and weighing 250-300 g, were
used. All of the following experimental protocols were approved by the
animal committee of Keio University School of Medicine, Tokyo, Japan.
Animals were anesthetized with pentobarbital sodium (50 mg/kg ip). The
trachea was cannulated and connected to a ventilator, then ventilated
at a tidal volume of 10 ml/kg and a respiratory rate of 60 breaths/min.
Lungs were exposed by median sternotomy, and blood was withdrawn from
the heart. The trachea was ligated at the level of one tidal volume
above functional residual capacity and fixed on a microscope stage in
the supine position. The main pulmonary artery and left atrium were
catheterized. Pulmonary arterial pressure was measured with a pressure
transducer (SEN-6102M; Nihon Koden, Tokyo, Japan) connected to the
pulmonary artery cannula and monitored continuously during the
experiment (AcqKnowledge III; BIOPAC Systems). Krebs-Henseleit solution
containing 3% albumin and autoerythrocytes was used as the perfusate
(hematocrit = 6.5 ± 0.5%). An extracorporeal membrane oxygenator
(Merasilox-S; Senkou-ikakougyou, Tokyo, Japan) was connected to the
perfused lung circuit and equilibrated with 21%
O2 and 5%
CO2. The lungs were perfused at a
rate of 10 ml/min with a peristaltic roller pump, and the perfusate
from the left atrium was allowed to collect in the reservoir. The
perfusate gas tension and pH were measured using a 1306 blood-gas
analyzer (Instrumentation Laboratory) at the beginning of and at
intervals during each experiment. The pH was maintained between 7.38 and 7.42. The lung was humidified, and the surface temperature was
maintained at 37 ± 0.5°C. The left lingula of the lung was
observed under an in vivo microscopic system (SO-MI, Sankei, Tokyo,
Japan), employing normal and fluorescence objective lenses (×10,
×20, ×40), connected to a closed-circuit video system. The
in vivo microscopic system has three lights. The first is a normal lamp
light (Techno Light KTS-150, Kenko, Tokyo, Japan), the second a xenon
lamp light (Nikon, Tokyo, Japan) for fluorescence imaging, and the
third a laser power supply (Omnichrome, Chino, CA) for confocal
imaging. The image was displayed with a high-sensitivity charge-coupled
device camera (TEC-470, Optronics) or a high-sensitivity intensified
imager (II) camera (EktaPro intensified imager, Kodak, San Diego, CA)
and color video monitor (PVM-1444Q, Sony, Tokyo, Japan). The image was
recorded on videotape with a tape recorder (SVQ-260, Sony). To
determine the diameters of arterioles, capillaries, and venules and to
analyze the high-speed movement of leukocytes and erythrocytes inside
vessels, a confocal laser scanning microscope (Yokokawa, Tokyo, Japan)
was used. Views of high-speed movements of the cells were displayed by
using the dynamic confocal laser scanning microscope with the II camera and stored in a high-speed video recorder system (EktaPro TR6,000 system, Kodak). Velocities of leukocytes and erythrocytes were recorded
at a rate of 250 frames/s using the high-speed video system. Leukocytes
that remained in the same internal portions of capillaries were
excluded from the speed analysis. Centerline blood flow velocity
(Vc) was
determined in arterioles and venules. The mean blood cell velocity
(Vmean) was
calculated from
Vmean = Vc /1.6. The
vessel diameters
(Dv) of
arterioles and venules were measured by processing the confocal video
images with a computer-assisted digital image-analyzing system (Apple
Quadra 840-AV/Image 1.58). The vessel wall shear rates ( Visualization of vessel networks, erythrocytes, and leukocytes.
To visualize vessel networks, we administered FITC-dextran (mol wt
145,000), at a final concentration of 0.015%, into the perfusion
circuit. Erythrocytes were labeled with FITC. Rat blood was diluted
with phosphate-buffered saline (PBS) and centrifuged. The erythrocyte
pellet was then diluted with PBS. FITC was added at a final
concentration of 0.1 mg/ml. After a 30-min incubation at 37°C, the
solution was centrifuged and diluted with 5 ml of PBS. Thereafter, 1 ml
of the dilute solution was administered into the perfusion circuit,
when necessary.
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
) were
calculated based on the definition for a Newtonian fluid: = 8(Vmean/Dv)
(in s
1), as described
elsewhere (24). To examine differences in the rolling behavior of
leukocytes between the pulmonary and mesenteric microvessels as
described in RESULTS, we followed the
original definition of rolling described by Atherton and Born (1, 2). The leukocytes that exhibited a caterpillar-like rotation along the
microvascular endothelium were defined as the rolling cells. In
addition, because some of previous works defined the rolling cells as
those temporarily interacting with vascular endothelium irrespective of
the presence of the caterpillar movement (12, 14), we have also
evaluated the rolling behavior based on this definition (see
RESULTS).
Experimental groups. We designed four experimental groups: 1) control, 2) rat IL-8, 3) anti-CD18, and 4) anti-ICAM-1. For the control group (n = 6), CFSE-labeled leukocytes and perfused rat lungs had no pretreatment. For the rat IL-8 group (n = 6), blood including CFSE-labeled leukocytes was incubated with 10 nM CINC/gro at 37°C for 10 min just before administration into the perfusion circuit. For the anti-CD18 group (n = 6), blood including CINC/gro-activated CFSE-labeled leukocytes (same as the rat IL-8 group) was treated with WT-3 for 30 min at room temperature at a final concentration of 50 µg/ml. The blood was then administered into the perfusion circuit. For the anti-ICAM-1 group (n = 4), 1A29 was administered into the perfusion circuit at a perfusate concentration of 10 µg/ml. After a 10-min perfusion period, blood including CINC/gro-activated CFSE-labeled leukocytes (same as the rat IL-8 group) was administered into the perfusion circuit. Separately, we conducted a series of experiments using isotype-matched mouse IgG samples as a control group for those treated with anti-CD18 or anti-ICAM-1 MAbs. CFSE-labeled leukocytes were stimulated with CINC/gro and incubated with mouse IgG at a final concentration of 50 µg/ml for 30 min. We also administered mouse IgG at a final concentration of 10 µg/ml over 10 min into the perfused rat lung circuit.
Roles of selectins in normal pulmonary leukocyte-endothelial interactions were examined by using a MAb 2H5 (n = 4), which recognizes SLeX and blocks its function to attenuate L-, P-, and E-selectin-mediated cell adhesion (26, 27). Because L- and P-selections might be involved, under our experimental conditions (L-selectin on leukocytes and P-selectin on pulmonary endothelial cells and the small population of platelets in the perfusate), 2H5 was added to the perfusate at a final concentration of 20 µg/ml, known to be sufficient to abolish selectin-dependent cell adhesion in vitro (27). 2H5 was also added to the leukocyte suspension to block the possible interaction of the neutrophil SLeX with P-selectin at the same MAb concentration. Blood including CFSE-labeled leukocytes was incubated with 2H5 for 30 min at room temperature, and the perfused rat lung was also treated with 2H5 for 10 min.Histological examination. A catheter was inserted into the main bronchus after each experiment, the duration of which was 30 min after injection of CFSE-labeled leukocytes. The perfused rat lung was then fixed with Formalin and embedded in paraffin. Six sections were cut at the same interval from the top to the bottom of the left lung and stained with hematoxylin-eosin. Differential intracapillary leukocyte counts were obtained at a magnification of ×1,000. In each section, 10 different microscopic fields selected at random were examined, and the density of leukocytes was expressed as numbers per single alveolus. The same sections served as samples for estimating cell differentials. Mononuclear cells in the circulation were distinguished as being inside the endothelium, whereas macrophages were outside the endothelium. We identified the capillary endothelium based on its location surrounding alveoli.
Distribution of ICAM-1 in pulmonary microvessels. Fluorescence-labeled 1A29 (4 µg/g body wt) was injected into an anesthetized ventilated rat via the femoral vein. Five minutes after the injection of fluorescence-labeled 1A29, the rat was prepared for perfusion. Nonbinding fluorescence-labeled 1A29 was washed out of the perfused rat lung. ICAM-1 labeled with fluorescence-labeled 1A29 was visualized with a confocal laser scanning microscope assisted by the image analyzer mentioned above. To confirm anatomic orientation of the pulmonary microvasculature, the proximal and distal landmarks such as arterioles and venules were recognized by injecting FITC-labeled red blood cells or dextran at the end of each experiment.
We also examined the ICAM-1 distribution using a conventional immunohistochemical approach. The lungs were fixed by intratracheal instillation of periodate-lysine-paraformaldehyde solution and embedded in optimal cutting temperature (OCT) compound (Miles, Elkhart, IN), then frozen in dry ice and acetone. Cryostat sections (6 µm each) were air dried for 1 h at room temperature. The sections were washed in PBS and incubated with 10% normal goat serum in PBS. To inhibit endogenous peroxidase activity, the sections were treated with methanol and 3% hydrogen peroxide for 20 min according to the method of Streefkerk (23). 1A29 antibodies were diluted 100-fold with PBS and absorbed with normal rat serum. Nonimmunized mouse serum was used instead of 1A29 as a negative control. These sera were layered on the section for 2 h at room temperature. Sections were incubated with peroxidase-labeled goat anti-mouse IgG (H + L) (Zymed, San Francisco, CA) for 30 min at room temperature and rinsed in PBS for 5 min three times. Then, labeled peroxidase was detected by reaction with 3,3'-diaminobenzidine tetrahydrochloride in 3% hydrogen peroxide tris(hydroxymethyl)aminomethane buffer for 4-10 min. Counterstaining of nuclei was performed with methyl green. Immunoreactivity for ICAM-1 was determined under a light microscope.In vivo adhesion assay of CFSE-labeled leukocytes in the mesenteric microcirculation. In separate sets of experiments, the behavior of CFSE-labeled leukocytes in the mesenteric venules (30-40 µm in diameter) was visually examined by injecting the suspension of CFSE-labeled leukocytes into recipient rats. Leukocytes were stained with CFSE according to the method described above. After centrifugation at 2,000 revolutions/min for 10 min, the leukocyte-rich fraction was collected and resuspended to give a final concentration of 5,000 cells/µl. The CFSE-stained leukocytes were divided into two groups: CINC/gro-prestimulated and unstimulated cells. The behavior of CFSE-labeled leukocytes in the postcapillary venules was visualized by intravital microscopy using a silicon intensified target camera (C-2400-08, Hamamatsu Photonics, Shizuoka, Japan) to monitor the erythrocyte velocity (Vr) as described elsewhere (25). Briefly, the ileocecal portion of the mesentery was opened and mounted on a plastic stage. After the 20-min stabilization period, 1 ml of the CINC/gro-stimulated or unstimulated leukocytes was injected for 2 min from the carotid artery while observing the mesenteric microcirculation under fluorescence microscopy with epi-illumination at 480 nm. In both groups, the leukocyte traffics in the mesentery were recorded for 30 min, and the influx of total fluorescent-labeled leukocytes was measured at 0, 10, 20, and 30 min after the cell injection (each experiment, n = 5). In other experiments (each experiment, n = 6), N-formyl-methionyl-leucyl-phenylalanine (FMLP) was superfused on the mesentery at a concentration of 100 nM to evoke venular adhesion of the CFSE-labeled cells. When the videotapes were replayed, the numbers of total, free flowing, and rolling CFSE-labeled leukocytes were estimated as described elsewhere (25). In addition, the frame-by-frame analysis also allowed us to determine the velocity of CFSE-labeled leukocytes (Vw). To examine differences in the leukocyte behavior between the CINC/gro-prestimulated and unstimulated cells, the ratio of Vw to Vr was estimated for individual leukocytes to establish histogram distribution of the Vw/Vr values. The leukocytes that stayed in the same portion of the venule for more than 30 s were defined as adherent cells.
Statistical analysis. The results are presented as means ± SE unless stated otherwise. All P values were determined using one-way analysis of variance (ANOVA) followed by the Scheffé-type multiple comparison test to detect differences among groups. A P value <0.05 was considered statistically significant.
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RESULTS |
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Abstract
Introduction
Methods
Results
Discussion
References
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Leukocyte behavior in pulmonary microcirculation. Mean pulmonary arterial pressure was 12 ± 2 (SD) mmHg and did not differ significantly among groups. Mean pulmonary arterial pressure was constant throughout the experiments. Three representative views of the periphery of the perfused rat lung are shown in Fig. 1. Alveoli were clearly visualized by light microscopy, whereas microvascular networks were delineated by confocal laser scanning microscopy. The mean diameters of arterioles and venules used for measurements were 15 and 18 µm, respectively, and exhibited no significant differences among the groups. The wall shear rates in arterioles and venules, under these conditions, are shown in Table 1. There are no statistically significant differences among the groups. The mean Vr in arterioles, capillaries, and venules of the control, rat IL-8, anti-CD18, and anti-ICAM-1 groups are shown in Fig. 2. There were no differences in mean arteriolar Vr among the groups. Mean Vr in capillaries and venules were also comparable among groups.
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Histological examination of leukocyte sequestration in pulmonary capillaries. The number of leukocytes, including polymorphonuclear neutrophils (PMNs) and mononuclear cells, in the periphery of the perfused lung in the rat IL-8 group was 2.70 ± 0.50/alveolus, which was increased as compared with that of the control group (0.35 ± 0.09/alveolus) (Table 3). The increased leukocyte numbers in the rat IL-8 group were partly attenuated by treatment with anti-CD18 and anti-ICAM-1 MAbs, whereas those in the anti-CD18 (0.91 ± 0.17/alveolus) and anti-ICAM-1 (1.20 ± 0.08/alveolus) groups were significantly elevated as compared with that in the control group. The differential ratios of capillary leukocytes are shown in Table 3. CINC/gro increased the proportion of PMNs sequestered in capillaries by 19.2 ± 3.4-fold as compared with that of the control group.
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Distribution of ICAM-1 in pulmonary microcirculation. The ICAM-1 distribution, as revealed by fluorescence-labeled 1A29 and the confocal laser scanning microscope, was observed mainly in capillary networks (Fig. 6A) and venules (Fig. 6B). There was only slight expression of ICAM-1 in arterioles. The arterioles and venules were confirmed by FITC-dextran and FITC-labeled red blood cells (Fig. 6C). There was no detectable fluorescence along vessels in the lung pretreated with FITC-labeled mouse IgG (Fig. 6D).
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Expression of adhesion molecules on leukocytes. Rat leukocytes were stimulated with 10 nM CINC/gro for 10 min to examine the surface expressions of SLeX, L-selectin, and CD18 on neutrophils and lymphocytes with a FACScan flow cytometry system (Fig. 8). In response to CINC/gro, L-selectin and SLeX were markedly downregulated, whereas CD18 was inversely upregulated on the surface of neutrophils. We confirmed that these changes elicited by CINC/gro occurred specifically on rat neutrophils but not on lymphocytes, supporting previous observations (18). These results suggest that, under the current experimental conditions, CINC/gro specifically stimulates neutrophils.
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Leukocyte behavior in mesenteric microcirculation.
In the mesentery, leukocytes exhibited characteristics different from
those observed in the lung preparation. In the observed mesenteric
microcirculation, the venular wall shear rates were between 300 and 500 s1, values no greater than
those measured in the perfused lung preparation. Figure
9 illustrates time history of the influx of
CFSE-labeled leukocytes in the mesenteric venules, showing no
significant difference between the two groups until the initial 10-min
recording period after the cell injection. However, the difference in
the cell influx became evident in a time-dependent manner, and the
disappearance rate of the CINC/gro-prestimulated leukocytes turned out
to be greater than that of the unstimulated leukocytes, presumably
because of entrapment of these prestimulated cells in other organs
(e.g., lung).
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The sequential multistep processes involving selectin-dependent rolling and integrin-dependent adhesion have been thought to serve as a central mechanism for stationary leukocyte interaction with endothelium in vivo and in vitro (6, 15, 22, 30). However, the present study first provides visible evidence showing specific leukocyte kinetics in the perfused lung ex vivo. Under physiological shear rates, prestimulated leukocytes lacking L-selectin can establish stationary leukocyte-endothelium interaction through ICAM-1-dependent and -independent mechanisms without undergoing the rolling behavior. At the same time, our observation actually demonstrated the possibility that the lung could allow circulating leukocytes to marginate in pulmonary microvessels irrespective of the presence of L-selectin, which was previously evaluated in L-selectin knockout mice by screening histochemical sections of the fixed lung samples (11).
There are several lines of evidence indicating that pulmonary leukocyte behavior is distinct from that in the mesenteric microcirculation, which is known to follow the multistep theory (30). First, the leukocyte rolling characterized by caterpillar-like movement was not observed in unstimulated leukocytes running through arterioles or venules in the lungs, whereas the same leukocytes rolled and adhered to mesenteric venules. This finding indicates that, under the current experimental conditions, a transient interaction between leukocytes and endothelium in the steady-state lung is quite different from that in the mesentery. Second, blockade of SLeX, a ligand for P-, E-, and L-selectins, did not alter the leukocyte behavior in the lung. Third, when activated, leukocytes were trapped mainly in pulmonary microvessels despite the absence of L-selectin. Finally, in the lung, the majority of leukocyte sequestration occurs in alveolar capillaries rather than in postcapillary venules, illustrating features quite different from those observed in mesenteric microvessels. Such a preferential accumulation in the capillaries was reported previously by Lien et al. (16), who studied C5a-induced pulmonary leukocyte sequestration. These results suggest that leukocyte accumulation in pulmonary microvessels involves mechanisms distinct from those in the mesenteric microvessels in that the pulmonary adhesion process is selectin independent even under physiological shear rates.
The present study provides visible evidence that capillary entrapment, but not venular adhesion, is a major mechanism underlying pulmonary leukocyte accumulation under the current experimental conditions. The processes of leukocyte entrapment involve specific patterns of interactions with microvascular endothelium including sudden arrest, plugging, and adhesion to capillary walls. In addition, the CD18-ICAM-1 interaction is likely to be attributable to the capillary entrapment, inasmuch as immunoneutralization of these molecules attenuated the density of leukocytes trapped in the alveolar capillaries. As was observed in mouse lungs (5), ICAM-1 was shown to be expressed abundantly in rat alveolar capillaries. The CINC/gro-stimulated neutrophils are thus in a position to utilize their upregulated CD18 molecules to facilitate specific binding to capillary endothelial ICAM-1.
On the other hand, another mechanism to be taken into account is capillary entrapment through CD18-ICAM-1 independent mechanisms, since anti-CD18 and anti-ICAM-1 MAbs attenuated only a portion of the CINC/gro-induced capillary leukocyte entrapment. Changes in leukocyte deformability induced by activation and mismatch of leukocyte-capillary diameters have been postulated to be the mechanism underlying CD18-independent leukocyte sequestration in pulmonary capillaries (9, 34). Because IL-8 is known to markedly change the intracellular actin polymerization in neutrophils and thereby increase cell stiffness (33), changes in viscoelastic properties of the neutrophil membrane may serve as a mechanism mediating capillary leukocyte sequestration. Collectively, our observations showing mechanical entrapment of activated leukocytes in pulmonary microvessels (Fig. 3) provide evidence that an alternative mechanism independent of adhesion molecules underlies pulmonary leukocyte accumulation.
Doerschuk (8) reported that CD18 does not mediate the initial sequestration of neutrophils but is required to keep the sequestered neutrophils in the lungs for more than a few minutes. These results are consistent with ours in view of the fact that tissue accumulation of leukocytes in the lung involves both CD18-dependent and -independent mechanisms, although there are several differences in the experimental protocols. Our observation was achieved by injecting a controlled number of CINC/gro-prestimulated leukocytes into the normoperfused lung. Accordingly, we could not observe the initial sequestration of neutrophils in the lung that is generally observed in response to the intravascular administration of stimuli such as zymosan-activated plasma into rabbits (8).
The capillary accumulation of stimulated leukocytes that occurs under physiological shear rates appears to be unique to the pulmonary microcirculation. In other organs such as the heart, brain, and skeletal muscles, the leukocyte entrapment in capillaries occurs only when the microvascular system is exposed to low-shear conditions elicited by hemorrhagic shock or ischemia (3, 7, 24). In addition, the current study has clinically important implications for understanding why the lung is a primary target of organ damage in endotoxemia and sepsis, in which a variety of cytokines including IL-8 are known to be released into the circulation (19, 21) and circulating neutrophils shed their L-selectin (21). Under these circumstances, such intravascular activation of neutrophils may cause redistribution of neutrophils in the marginating pool, i.e., demargination of neutrophils from the extrapulmonary microcirculatory system (e.g., intestinal microcirculation) and their selectin-independent entrapment in the lung microcirculation. When systemic hemodynamics are markedly impaired during sepsis, further leukocyte entrapment may occur in alveolar capillaries, thereby leading to lung injury. Although the full picture of the leukocyte accumulation mechanisms operating in the lung microcirculation under disease conditions has yet to be elucidated, the present results shed light on the effectiveness and limitations of antiadhesion therapy, which may induce leukocyte accumulation and subsequent lung injury.
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ACKNOWLEDGEMENTS |
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We thank Dr. K. Watanabe (Institute of Cytosignal Research, Tokyo, Japan) and Dr. M. Miyasaka (Dept. of Bioregulation, Osaka University Medical School, Biomedical Research Center, Osaka, Japan) for gifts of CINC/gro and WT-3, 1A29, and HRL-4, respectively. We also appreciate Dr. T. Tamatani (Pharmaceutical Basic Research Laboratories, Japan Tobacco Inc., Yokohama, Japan) for providing 2H5.
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FOOTNOTES |
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Address for reprint requests: K. Yamaguchi, Dept. of Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan.
Received 6 August 1996; accepted in final form 7 July 1997.
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Abstract
Introduction
Methods
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Discussion
References
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and 
,
Continuous and discontinuous patterns, respectively, in the control
group (A); 
, 
, 
: discontinuous patterns of three
different leukocytes in rat IL-8 group (B).
P < 0.05 and 
) and CINC/gro(+) indicate data collected from animals
injected with CINC/gro-unstimulated and -prestimulated cells,
respectively. Influx is expressed as number of total (free-flowing and
rolling) fluorescent-labeled leukocytes passing through a mesenteric
venule for 1 min at 0, 10-, 20-, and 30-min observation period after
completing cell injection. Note that there was no difference in influx
of fluorescent-labeled leukocytes between CINC/gro-unstimulated and
-prestimulated cells during initial 10-min period. Values are means ± SD of 5 separate experiments in both groups.
* P < 0.05 and
** P < 0.01 between groups.
