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Departments of 1Medicine and 2Biochemistry, School of Medicine, Keio University, Tokyo; 3Department of Medicine, Kitasato Institute Hospital, Tokyo; 4Pharmaceutical Frontier Research Laboratories, JT Incorporated, Yokohama; and 5Biochemical Department, Sankei Corporation, Tokyo, Japan
Submitted 22 August 2005 ; accepted in final form 28 December 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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neutrophil; lymphocyte; rolling; adhesion
The accumulation of neutrophils in the lung tissue is initially mediated through increased adhesion of neutrophils to endothelial cells of the inflamed pulmonary microvasculature, and this process requires the upregulation of various adhesion molecules expressed on the surface of pulmonary microvessel endothelial cells as well as circulating neutrophils (18, 22). Despite the importance of augmented expression of adhesion molecules for initiating neutrophil-evoked lung injury, there has been little study shedding light on this issue in lung models of VILI, except that reported by Yiming et al. (37). These authors demonstrate that P-selectin expression on endothelial cells of the pulmonary vasculature was enhanced at an early stage after commencing artificial ventilation with a high tidal volume, followed by neutrophil accumulation. Unfortunately, however, they did not analyze other important adhesion molecules mediating neutrophil adhesion to pulmonary endothelial cells, such as ICAM-1 and VCAM-1. Furthermore, the relative contribution of each hierarchy of microvessels, including arterioles, venules, and capillaries, to neutrophil sequestration to the pulmonary microcirculation has never been examined in a VILI model, although this approach is essential for deepening our understanding of the early pathogenesis of VILI.
In addition to neutrophil infiltration in the lung tissue, lymphocyte accumulation and activation may occur at sites of inflammation in VILI. However, there has been no reliable study analyzing the contribution of lymphocytes to the initiation and/or aggravation of VILI. Lymphocytes may have a number of functions, including immune response, release of various cytokines, and cytotoxicity (36), all of which are expected to play a pivotal role in the development and pathogenesis of VILI. Although the recruitment of lymphocytes to inflammatory sites in systemic organs and tissues has been shown to be mediated in part through an adhesive mechanism involving ICAM-1- and/or VCAM-1-dependent pathways (2, 34), the issue of whether the same holds true for lymphocyte recruitment to the microcirculation of the diseased lung has not been extensively addressed.
To elucidate the adhesive mechanism mediating the accumulation of neutrophils and lymphocytes in arterioles, venules, or capillaries in lungs injured by sustained artificial mechanical ventilation, we analyzed the following issues: 1) differential expression of various classes of adhesion molecules, including ICAM-1, VCAM-1, and P-selection, along arteriolar, venular, and capillary walls in a rat model of VILI; 2) measurements of the dynamic behavior of polymorphonuclear cells (PMN: mainly composed of neutrophils) within arterioles, venules, and capillaries in the lung isolated from an animal exposed to prolonged ventilation by means of recently developed real-time confocal laser fluorescence microscopy in the presence or absence of MAb to each endothelial adhesion molecule; and 3) confocal microscopic measurements of the dynamic behavior of mononuclear cells (MN: mainly consisting of lymphocytes) in each microvessel of the isolated lung with VILI, with and without MAb, to the respective adhesion molecules.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Preparation of fluorescein-labeled PMN and MN. We aspirated whole blood from the left ventricle of normal donor rats, in which 28 ± 9% of leukocytes were PMN and the remainder were MN (lymphocytes: 72 ± 9%). We isolated PMN and MN from whole blood aspirated from donor rats by applying a classic Ficoll density gradient procedure adapted for either rat PMN (specific gravity: 1.112) or MN (specific gravity: 1.090) (20). Both cell layers were labeled with carboxyfluorescein diacetate succinimidyl ester (CFDASE). This fluorescence precursor yields carboxyfluorescein succinimidyl ester (CFSE) in leukocytes but not in erythrocytes (27). After 30-min incubation of each leukocyte layer with CFDASE in vitro, the final solution was added to the perfusion reservoir connected to the isolated perfused lung system (see below). Preliminary experiments confirmed that the layer of PMN separated by the Ficoll method was mostly composed of neutrophils (96 ± 0.8%). In the layer of MN obtained by qualitatively the same method as applied for PMN, most of the blood cells were found to be lymphocytes (99 ± 0.5%). Leukocytes (PMN and MN) and platelets stained with CFSE were easily distinguishable during microscopic observation because of their distinctive difference in size.
Preparation of isolated perfused lungs.
The animals with 6-h artificial ventilation were not anesthetized again, but the control animals were anesthetized with pentobarbital (50 mg/kg, intraperitoneally). These animals were artificially ventilated with room air for only the short time necessary to prepare the isolated perfused lung system. After median sternotomy was performed, animals were killed by exsanguination, and cannulas were inserted into the pulmonary artery and left atrium. Both cannulas were secured with ligatures. A ligature was placed around the aorta to prevent loss of perfusate into the systemic circulation. Isolated lungs prepared from the control, low tidal, and high tidal groups were fixed on a microscope stage in the supine position and perfused at a constant flow rate of 10 ml/min with physiological solution containing a small amount of whole blood 
10 min after exsanguination. Modified Krebs-Henseleit solution was used as the perfusate, and 3% bovine serum albumin was added to maintain isoosmotic pressure. Perfusate hematocrit was adjusted to 3.2 ± 0.8% by the addition of fresh blood obtained from donor rats, which were pretreated with 5 mg/kg heparin. Because the total volume of perfusate was adjusted to 100 ml, heparin concentration in the circulation system was 6.5 µg/ml. To avoid movement caused by artificial ventilation, the trachea was ligated at the end-inspiratory point, and gas exchange was maintained with an extracorporeal membrane oxygenator (ECMO, Merasilox-S; Senko, Tokyo, Japan). A gas mixture containing 21% O2 and 5% CO2 in N2 was used as the gas flowing into the ECMO, resulting in perfusate PO2, PCO2, and pH of 142 ± 6 Torr, 37 ± 2 Torr, and 7.41 ± 0.03, respectively. A warmed and humidified gas mixture containing the same composition of gases as those used for the ECMO was continuously supplied to the lung surface to maintain a temperature of 37 ± 0.2°C and to avoid desiccation of the lung surface. Pulmonary arterial pressure was continuously monitored by force displacement of a pressure transducer (TP-400T; Nihon Kohden, Tokyo, Japan).
Experimental protocols. To investigate the effects of various adhesion molecules on the microcirculatory behavior of leukocytes in the early stage of VILI, we designed six experiments for both CFSE-stained PMN and CFSE-stained MN: 1) Control group (PMN n = 6, MN n = 7): animals breathed spontaneously without a ventilator and anesthesia. 2) Low tidal group (PMN n = 5, MN n = 5): animals were ventilated with a tidal volume of 7.5 ml/kg for 6 h. 3) High tidal group (PMN n = 9, MN n = 9): animals were ventilated with a tidal volume of 15 ml/kg for 6 h. 4) ICAM-1 group (PMN n = 6, MN n = 8): MAb against rat ICAM-1 (1A29; donated by Dr. Miyasaka, Osaka University Medical School, Osaka, Japan) was administered to isolated perfused lungs prepared from animals with VILI. Perfusate concentration of 1A29 was maintained at 20 µg/ml. After 10-min perfusion with 1A29, the microcirculatory behavior of PMN or MN was investigated. The efficiency of 1A29 in blocking rat ICAM-1 was extensively discussed by Tamatani and Miyasaka (29). 5) VCAM-1 group (PMN n = 5, MN n = 7): MAb against rat VCAM-1 (MR106; donated by Dr. Yagita, Juntendo University, School of Medicine) was administered at a final concentration of 20 µg/ml into perfused lungs prepared from the animals with high tidal volume ventilation. Ten minutes after the addition of MR106, PMN or MN kinetics was observed. 6) P-selectin group (PMN n = 6, MN n = 5): MAb against rat P-selectin (s789G) was administered at a final concentration of 20 µg/ml into perfused lungs prepared from the animals with high tidal volume ventilation. Ten minutes later, PMN or MN kinetics was examined. The specificity of s789G in inhibiting rat P-selectin is described in detail elsewhere (11).
Observation of microvascular cell kinetics by confocal fluorescence microscopy. We used a real-time confocal laser scanning fluorescence optical microscope (CSU10; Yokokawa, Tokyo, Japan) incorporating a high-speed video analysis system (35). The reflected light or fluorescent emission from the specimen was imaged onto a high-sensitivity charge-coupled device (CCD) camera with an image intensifier (VSG; Kodak, San Diego, CA). By incorporating an excitation wave length of 488 nm emitted from a low-power air-cooled argon laser (532-BSA04; Omnichrome, Cino, CA), the present confocal system allowed us to obtain apparently instantaneous images at 1,000 frames/s. The final magnifying power of our system reachedx484 with a x20 objective. We obtained confocal images of microvessels on the plane that was in focus at 5–25 µm distance from the surface of the left upper lung. We registered confocal images at a rate of 125 frames/s with a high-speed digital video analysis system (1000 Processor; Kodak, Tokyo, Japan) connected to the image-intensified CCD camera. All images were stored on the hard disk of a personal computer.
To examine the erythrocyte velocity (Vr) in the same microvessel used for assessment of PMN or MN behavior, erythrocytes were stained with fluorescein isothiocyanate (FITC; Sigma, St. Louis, MO). Fresh rat blood was centrifuged at 1,000 rpm for 5 min, and the buffy coat was discarded. The packed erythrocyte solution was diluted with PBS, and FITC was added to give a final concentration of 0.1 mg/ml. This solution was added to the reservoir after PMN or MN kinetics had been measured, and then erythrocyte kinetics was determined, as well.
To examine the architecture of microvessels in which leukocyte and erythrocyte behavior had been analyzed, 200 µl of 5% FITC-dextran with a molecular weight of 145,000 (Sigma) were added to the reservoir. The microvessel from which fluorescein-labeled blood cells entered the capillary network was defined as the arteriole, whereas the microvessel into which blood cells flowed from the capillary network was taken to be the venule.
Quantitative analysis of cell kinetics in arterioles, venules, and capillaries.
The highest value of Vr was assumed to be equal to the centerline velocity (Vmax) in a given arteriole or venule. Vmax was used to estimate mean fluid flow (Vmean) in each microvessel, where Vmean = Vmax/1.6 (8). Wall shear rate (
) was estimated from the equation: = 8 (Vmean/D), where D represents vessel diameter (23). An adherent leukocyte in an arteriole or venule was defined as a cell that was firmly attached to the vascular endothelium and did not move during the observation period. Rolling leukocytes in arterioles and venules were defined as cells transiently interacting with the vascular endothelium and thus traveling much more slowly than centerline erythrocytes. We identified these cells by applying the velocity criterion proposed by Gaehtgens and colleagues (8), who calculated the critical velocity (Vcrit) of a freely flowing cell traveling close to, but not adhering to, the microvascular wall. Vcrit is defined as Vmean(2–
), where is the ratio of the leukocyte diameter to the microvessel diameter. Any cell flowing at a velocity below Vcrit was assumed to be slowed by the interaction with the vascular endothelium and defined as a rolling cell.
To quantify the cell transit in capillaries, we classified the observed leukocytes into two categories: 1) cells moving smoothly without interruption and 2) impeded cells stopping for >20 ms within a capillary segment, thus including the cells with transient entrapment and those with sustained arrest in capillaries during the observation period.
Intravital determination of ICAM-1, VCAM-1, and P-selectin expression along microvessel walls. We examined the expression of ICAM-1, VCAM-1, and P-selectin along microvessel walls by applying the intravital fluorescence microscopic method (n = 3 for each). This method allows reliable separation of arterioles from venules in association with quantitative determination of each adhesion molecule expressed along microvessel walls. In brief, 4 µg/g body wt of 1A29 (MAb against ICAM-1), MR106 (MAb against VCAM-1), or s789G (MAb against P-selectin) were injected into the pulmonary artery via the perfusion catheter. Ten minutes later, 0.5 ml of FITC-labeled anti-mouse IgG antibody (Sigma), the secondary antibody to each MAb, was injected into an isolated perfused lung. Subsequently, flow was stopped for 15 min to allow conjugation of a given adhesion molecule on the vascular endothelium, its MAb, and FITC-IgG antibody. The final complex of the adhesion molecule bound to its MAb and FITC-IgG antibody was examined under a confocal scanning fluorescence microscope and displayed on a high-sensitivity CCD camera (TEC-470; Optronics, Goleta, CA). As the MAb used in the present study consisted of mouse IgG, IgG raised in mice (4 µg/g body wt, Sigma) and FITC-labeled anti-mouse IgG antibody were used as negative controls. Arterioles, venules, and capillaries were distinguished by administering both FITC-dextran and FITC-labeled erythrocytes at the end of each measurement. We measured the complex-elicited fluorescence intensity along microvessel walls by processing a confocal image with a digital image-analyzing system (Quadra 840AV/Image 1.58; Apple, Cupertino, CA).
Determination of ICAM-1, VCAM-1, and P-selectin expression by immunohistochemical staining. The lungs with high tidal volume ventilation were fixed by administration of periodate-lysine-paraformaldehyde solution through the trachea, embedded in optimum cutting temperature compound (Miles, Elkhart, IN), and then frozen in dry ice and acetone (n = 5). Frozen sections 4 µm in thickness were cut on a cryostat and fixed on slides in cold acetone for 10 min and overnight at –20°C. These sections were washed with PBS, and nonspecific staining was inhibited by incubation with 10% normal pig serum (COSMO BIO, Tokyo, Japan) in PBS for 20 min at room temperature. The sections were then incubated with anti-ICAM-1 (M-19; Santa Cruz Biotechnology, Santa Cruz, CA), anti-VCAM-1 (C-19, Santa Cruz), or anti-P-selectin antibody (C-20, Santa Cruz) diluted 10-fold with PBS, for 1 h at room temperature. After these sections were rinsed with PBS, they were incubated with a 100-fold dilution of secondary antibody (anti-goat IgG-horseradish peroxidase, Santa Cruz) for 1 h at room temperature. After they were rinsed with PBS, they were incubated with 20 mg 3,3'-diaminobenzidine tetrahydrochloride and 20 µl of 30% hydrogen peroxide in 100 ml of PBS for 2 min at room temperature. After washing them in water, we performed counterstaining of nuclei with methyl green. Immunoreactivity for ICAM-1, VCAM-1, or P-selectin in each lung section was determined with a light microscope. In total, 30 sections obtained from each of the upper, middle, and lower lung fields of five different animals were used for immunohistochemical assessment of ICAM-1, VCAM-1, and P-selectin expression in the control, low tidal volume-ventilated and VILI lungs, respectively.
Histological examination and BAL. The lungs harvested from control and low and high tidal volume-ventilated rats were inflated with formalin at a pressure of 30 cmH2O for 48 h (n = 5 for each). Six sections, cut at identical intervals from the apex to the base of the left lung, were embedded in paraffin and stained with hematoxylin-eosin. In each section, 10 fields were randomly chosen, and leukocytes localized within the alveolar septa in which the capillary network is located were counted at a magnification of x1,000 with an oil immersion lens. The density of leukocytes in the septa was expressed as the number per single alveolus. The same lung sections were used for estimating leukocyte differential counts, i.e., the distinction between PMN and MN. The histological samples obtained from each animal were evaluated in a double-blinded fashion by two doctors.
BAL was carried out by washing the lung with 3 ml pf phosphate-buffered saline twice. The number of total cells and the differential cell counts were determined on Diff-Quick-stained preparations (Kokusai Shiyaku, Kobe, Japan).
Statistical analysis. The results are presented as means ± SD. Significant differences in the frequency of rolling cells (PMN and MN), their capillary entrapment, and their accumulation in alveolar septa among experimental groups (i.e., Control, Low, High, High+anti-ICAM-Ab, High+VCAM-Ab, and High+P-selectin-Ab groups) were determined by applying one-way analysis of variance (ANOVA) followed by Scheffé's multiple-comparison analysis. The values obtained for arterioles, venules, and capillaries in respective experimental conditions were statistically examined by two-way ANOVA together with Scheffé's analysis for multiple comparisons. A P value <0.05 was taken to be statistically significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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In the histological analysis, the high tidal group showed no serious tissue injury but slightly thickened alveolar walls and significant leukocyte infiltration into perivascular and peribronchiolar regions as well as alveolar spaces. The number of PMN accumulated within the alveolar septa in the high tidal group averaged 0.32 ± 0.19/alveolus, being much higher than that in the control group (0.11 ± 0.09/alveolus) and the low tidal group (0.15 ± 0.12/alveolus) (Fig. 1D). Similarly, the number of MN accumulated within the alveolar septa in the high tidal group (0.09 ± 0.01) was significantly higher than that in the control (0.04 ± 0.10, P < 0.05) and low tidal groups (0.03 ± 0.05, P < 0.05) (Fig. 1D).
Expression of ICAM-1, VCAM-1, and P-selectin in lungs evaluated by immunohistochemical staining. Alveolar walls in the lungs of the control and low tidal groups showed faint but positive immunoreactivity for ICAM-1. ICAM-1 expression was also detected along venules and capillaries, but not along arterioles, in the lungs of the control and low tidal groups (Fig. 2, A and B). In high tidal volume-ventilated lungs, ICAM-1 expression was appreciably enhanced along the alveolar walls as well as venular and capillary segments. Furthermore, ICAM-1 expression was upregulated and discernible along the arteriolar walls in high tidal volume-ventilated lungs (Fig. 2C).
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Expression of ICAM-1, VCAM-1, and P-selectin along microvessel walls evaluated by intravital fluorescence microscopy. In negative control experiments involving the administration of mouse IgG and FITC-labeled anti-mouse IgG antibody, no fluorescence was detectable along microvascular walls in any experimental group. Intravital confocal fluorescence analysis confirmed that ICAM-1 was expressed along venular and capillary walls, but not along arteriolar walls, in the control group (Fig. 2, D and E). On the other hand, ICAM-1 was significantly upregulated not only along venular and capillary walls, but also along arteriolar walls, during the development of VILI (Fig. 2, F and G).
VCAM-1 was not detectable along any microvessels in the control group (Fig. 2K). VCAM-1 was upregulated in both venular and capillary walls, but not in arteriolar walls, in the high tidal group (Fig. 2, L and M).
In the control group, P-selectin was not detectable in any microvessels (Fig. 2Q). Although P-selectin was appreciably upregulated along venular walls in the high tidal group, P-selectin expression was not evident along arteriolar and capillary walls (Fig. 2, R and S).
Compared with immunohistochemical study, intravital fluorescence microscopy has some advantages, especially in the identification of microvessels along which a given adhesion molecule is expressed. The intravital microscopic method allows examination of adhesion molecule expression under conditions in which microcirculatory flow is maintained. Therefore, arterioles and venules are easily identified from the difference in their flow directions. However, the sensitivity in detecting the expression of each adhesion molecule is expected to be relatively low with the intravital microscopic method compared with immunohistochemical staining. On the basis of these facts, we classified the degree of expression of each adhesion molecule into two categories: strong and faint enhancement. Strong enhancement was defined as upregulation of the adhesion molecule confirmed by both intravital microscopy and immunohistochemical study, whereas faint enhancement was defined as upregulation detected by immunohistochemical study only (Table 1). According to this classification, ICAM-1 expression was shown to be strongly enhanced along all microvessels, including arterioles, venules, and capillaries in high tidal volume-ventilated lungs. On the other hand, VCAM-1 expression was strongly enhanced along venules and capillaries but faintly enhanced along arterioles in VILI lungs. P-selectin expression in high tidal volume-ventilated lungs was strongly enhanced only in venules but faintly enhanced in both arterioles and capillaries.
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Vmean in arterioles was 0.92 ± 0.36 mm/s in the control group, 1.08 ± 0.75 mm/s in the low tidal group, and 0.93 ± 0.57 mm/s in the high tidal group, corresponding to a wall shear rate of 395, 419, and 394 s–1, respectively. Venular Vmean was 1.03 ± 0.48 mm/s in the control group, 1.12 ± 0.57 mm/s in the low tidal group, and 1.37 ± 0.61 mm/s in the high tidal group, yielding a wall shear rate of 379, 464, and 523 s–1, respectively. The values of Vmean and shear rates in arterioles were not significantly different among the control group, the low tidal group, and the high tidal group. Similarly, both values in venules among three groups were not significantly different. Vmean and wall shear rate were not influenced by the addition of any MAb. Vmean as well as wall shear rates tended to be lower in arterioles than those in venules, but statistically not significant.
We found no PMN adhering firmly to the arteriolar and venular walls under any experimental condition. In the high tidal group, the frequency of rolling PMN was much higher not only in venules (25.4 ± 14.8%), but also in arterioles (34.6 ± 15.2%), than in the control group (venule: 8.1 ± 11.6% vs. VILI, P < 0.05, arteriole: 11.8 ± 13.1% vs. High, P < 0.05) and in the low tidal group (venule: 5.5 ± 11.1% vs. High, P < 0.05, arteriole: 15.5 ± 8.7% vs. High, P < 0.05) (Fig. 3A). The augmentation of PMN rolling along the venular wall in high tidal volume-ventilated lungs was clearly inhibited by MAb against ICAM-1, VCAM-1, or P-selectin (Fig. 3A). Although PMN rolling along the arteriolar wall in the high tidal group was significantly inhibited by either anti-ICAM-1 MAb or anti-VCAM-1 MAb, it was not inhibited by the administration of anti-P-selectin MAb (Fig. 3A).
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The number of MN entrapped in capillaries was found to be significantly higher in the high tidal group (47.8 ± 19.3%) than in both the control (27.3 ± 12.2% vs. High, P < 0.05) and low tidal groups (24.1 ± 18.3% vs. High, P < 0.05). The enhanced entrapment of MN in capillary segments was effectively inhibited by MAb against ICAM-1 or VCAM-1 (Fig. 4B).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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for 4–10 h. Bhattacharya et al. (3) reported that high tidal ventilation upregulated P-selectin on the fresh lung endothelial cells obtained from the rats exposed to high tidal volume ventilation. These findings are highly consistent with those observed in the present study in which neither immunohistochemical study nor intravital fluorescence microscopy detected significant expression of VCAM-1 and P-selectin along any microvessels in control lungs (Fig. 2). In lungs with VILI, the expression of ICAM-1, VCAM-1, and P-selectin was found to be faintly or strongly enhanced along all the microvessels analyzed (Figs. 2–5 and Table 1). To our knowledge, there has been no authentic study confirming the upregulation of these adhesion molecules along arteriolar segments in the pulmonary microcirculation. We did not obtain evidence of upregulation of ICAM-1 along arteriolar walls in hyperoxia-injured lungs (18) or in bleomycin-induced fibrotic lungs (22) by applying the same method as used for the present analysis. On the basis of these facts, we consider that enhanced expression of ICAM-1, and probably VCAM-1 and P-selectin, along arteriolar walls is a unique event occurring in lungs injured by sustained artificial ventilation. The reason why VILI specifically enhances adhesion molecule expression in pulmonary arterioles, which is not seen in other models of lung injury, is not clear, but we speculate that one of the reasons is the stimuli by VILI is a mechanical stress and stronger than other stimuli such as hyperoxia or endotoxin. Therefore, further study is necessary to elucidate the actual cause of VILI-evoked upregulation of various adhesion molecules along arteriolar walls in the lung.
There is some room for doubt about the significance of endothelial VCAM-1 in modulating PMN kinetics along microvessels in diseased lungs, because of the uncertainties concerning the expression of its counterpart ligand, very late antigen-4 (VLA-4), on the PMN surface. VLA-4 has been considered to be mainly expressed on the mononuclear cell surface. Rieu et al. (21) as well as Lund-Johansen and Terstappen (14) reported that VLA-4 was not detected on the PMN surface, whereas Ibbotson et al. (9) showed that VLA-4 could be mobilized from intracellular granules to the plasma membrane in human blood PMN upon cell activation or during transendothelial migration. In addition, Shang and Issekutz (24) reported that VLA-4 was expressed on the surface of C5a-activated PMN, thus mediating ICAM-1-independent migration of PMN through the vascular endothelium to lung connective tissue. The integrin VLA-9, also expressed on neutrophils, is an alterative receptor for VCAM-1 (30). Although we could not directly measure the levels of VLA-4 and VLA-9 expression on the PMN surface, we believe that the interactions between endothelial VCAM-1 and PMN VLA-4 and VLA-9 are important pathways causing the abnormal microvascular kinetics of leukocytes, including MN and PMN in high tidal volume-ventilated lungs. This is because inhibition of VCAM-1 improved the microvascular rolling of MN and PMN in high tidal volume-ventilated lungs (Fig. 3).
There is no doubt about the importance of endothelial P-selectin in the mediation of abnormal leukocyte kinetics (PMN and MN) in pulmonary microvessels, especially in venules and capillaries, in diseased lungs. Bless et al. (4) demonstrated that, in the cobra venom factor model, P-selectin was upregulated along venular and capillary walls within 1 h, followed by its sustained expression for the next 7 h. Furthermore, P-selectin was confirmed to play a vital role in the development and pathogenesis of various types of lung injury (12, 16, 17). However, to our knowledge, no significant role of enhanced arteriolar P-selectin in evoking lung injury has been conclusively demonstrated. In the present study, we therefore attempted to clarify the importance of P-selectin expressed along arteriolar walls in addition to venular walls and capillary segments in the initial phase of lung injury caused by sustained artificial ventilation.
Importance of adhesion molecules modulating PMN kinetics in high tidal volume-ventilated lungs. Augmented PMN rolling along postcapillary venules in high tidal volume-ventilated lungs was significantly suppressed by MAb against ICAM-1, VCAM-1, or P-selectin (Fig. 3), indicating that these adhesion molecules are equally important for inducing venular PMN rolling in ventilator-associated lung injury. The direct effect of VILI on the adhesion molecule expressions of white blood cells is unclear. Ohta and coworkers (19) reported that in rat lungs receiving high tidal ventilation, the expression of Mac-1 and ICAM-1 on neutrophils and macrophages increased significantly more than in the low tidal ventilation. On the other hand, Choudhury and coworkers (7) showed in an in vivo mouse model of VILI that PMN sequestration in injurious ventilation was markedly inhibited by administration of anti-L-selectin antibody, but not by anti-CD18 antibody, suggesting that this phenomenon is mediated by L-selectin-dependent but CD18-independent mechanisms. Interestingly, however, no rolling PMN qualitatively similar to those seen in systemic venules appeared; i.e., rolling leukocytes in systemic venules frequently show caterpillar-like movement (32), whereas rolling PMN observed in pulmonary venules revealed smooth movement with a significant reduction in velocity compared with that of erythrocytes flowing in the central part of the microvessel. Furthermore, we should emphasize the fact that firm tethering of PMN along venular walls was not observed under any experimental condition in both intact and VILI preparations of our experimental system. These findings are in radical contradiction to the sequential two-step theory of endothelium-leukocyte interaction developed to explain leukocyte rolling and subsequent firm adhesion to vessel walls in the systemic microcirculation (6, 13, 32). Several lines of evidence based on observations in the systemic microcirculation showed that the selectin family such as P-selectin (endothelial surface) and/or L-selectin (leukocyte surface) would initiate cell rolling along microvessels (especially venules) as the first step, leading to firm adhesion mediated by the immunoglobulin superfamily such as ICAM-1 expressed on the microvascular endothelium as the second step (6). Our findings suggest that the second step of firm adhesion of PMN to endothelial cells is lacking or very weak in pulmonary venules, and only the rolling step of PMN plays a role in promoting the venular sequestration of these cells in diseased lungs. A further interesting point is that, besides P-selectin, both ICAM-1 and VCAM-1, which are adhesion molecules inducing firm adhesion between PMN and endothelial cells in systemic venules, act to cause PMN rolling in pulmonary venules.
In arterioles, augmented PMN rolling in high tidal volume-ventilated lungs was appreciably improved by the inhibition of endothelial ICAM-1 or VCAM-1 but not by the inhibition of P-selectin (Fig. 3). Although these findings are somewhat different from those observed for venules, in which all three adhesion molecules contribute equally to PMN rolling, they suggest that rolling in arterioles is as important as that in venules for PMN sequestration into the pulmonary microcirculation in high tidal volume-ventilated lungs. Again, we would like to emphasize the fact that ICAM-1- and VCAM-1-dependent, but P-selectin-independent, arteriolar PMN rolling, which is expected to reflect the initial stage of tissue injury, should be taken as a phenomenon unique to ventilator-associated lung injury.
The importance of various adhesion molecules expressed along capillary segments seems to differ qualitatively from that along venular or arteriolar walls, because we found that only ICAM-1 contributed to the enhanced capillary entrapment of PMN in high tidal volume-ventilated lungs (Fig. 4). The pathological significance of either VCAM-1 or P-selectin expression in abnormal PMN kinetics in capillaries with high tidal volume ventilation is not clear at present. Further study is necessary to demonstrate their exact role. Combining the findings on PMN kinetics observed for arterioles, venules, and capillaries, we can conclude that the contribution of different classes of adhesion molecules to PMN kinetics in the pulmonary microcirculation is vessel type specific (Table 2). Furthermore, besides the cell entrapment in capillary segments, PMN accumulation in the pulmonary microcirculation in ventilator-associated lung injury appears to be preferentially mediated via rolling, but not via firm adhesion, in both arterioles and venules.
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To summarize the findings obtained from lungs with VILI (Tables 1 and 2): the expression of endothelial adhesion molecules differs quantitatively between different microvessels; i.e., ICAM-1 expression is strongly enhanced along all microvascular walls, including arterioles, venules, and capillaries, whereas VCAM-1 is strongly upregulated in venules and capillaries but weakly upregulated in arterioles. P-selectin expression is significantly enhanced along venular walls but weakly enhanced along arteriolar and capillary walls. Consistent with the findings of adhesion molecule expression, the abnormal cell kinetics of both PMN and MN is importantly mediated via an ICAM-1-dependent pathway in all microvessels, suggesting that ICAM-1 inhibition could play a pivotal role as an effective therapeutic strategy in preventing the development of VILI. Furthermore, we would like to stress the fact that ICAM-1- and VCAM-1-dependent arteriolar rolling of PMN and MN appears to function as a specific mechanism for the accumulation of these inflammatory cells in the pulmonary microcirculation in the early stage of VILI.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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  A. Moldobaeva, J. Jenkins, and E. Wagner Effects of distension on airway inflammation and venular P-selectin expression Am J Physiol Lung Cell Mol Physiol, November 1, 2008; 295(5): L941 - L948. [Abstract] [Full Text] [PDF]
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 W. M. Kuebler, K. Parthasarathi, J. Lindert, and J. Bhattacharya Real-time lung microscopy J Appl Physiol, March 1, 2007; 102(3): 1255 - 1264. [Abstract] [Full Text] [PDF]
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