Ann Thorac Surg 2000;69:402-407
© 2000 The Society of Thoracic Surgeons
Original Articles
Superoxide possibly produced in endothelial cells mediates the neutrophil-induced lung injury
Tatsuo Tanita, MDa,
Chun Song, MDa,
Hiroshi Kubo, MDa,
Yasushi Hoshikawa, MDa,
Shinsaku Ueda, MDa,
Shigefumi Fujimura, MDa
a Department of Thoracic Surgery, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan
Address reprint requests to Dr Tanita, Department of Thoracic Surgery, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryomachi, Aoba-ku, Sendai 980-8575, Japan
e-mail: tanita{at}idac.tohoku.ac.jp
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Abstract
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Background. The mechanism by which stimulated neutrophils (polymorphonuclear leukocytes [PMNs]) damage pulmonary vascular endothelium was investigated.
Methods. The ability of unstimulated and mechanically stimulated PMNs to adhere to pulmonary endothelial cells and, thereby, alter pulmonary vascular permeability was tested. Each series was conducted on 6 rats. To stimulate PMNs, they were agitated gently in a glass vial for 10 seconds.
Results. Perfusing lungs with the stimulated PMNs elicited a fivefold increase in permeability compared with lungs perfused with the unstimulated cells. This increase in permeability was blocked completely by preincubation of stimulated PMNs with CD18 monoclonal antibody. This increase in permeability was also blocked completely by superoxide dismutase (SOD) or the xanthine oxidase (XO) inhibitor allopurinol. Pulmonary vascular hemodynamics were unaffected by any treatment protocol. The accumulation of stimulated PMNs within the lungs was not inhibited by SOD but was partially blocked by allopurinol.
Conclusions. These findings suggest that stimulated PMN-induced increases in pulmonary vascular filtration resulted from endothelial cell injury caused by superoxide anion possibly generated by XO, exclusively present in the endothelial cells.
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Introduction
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Acute respiratory distress syndrome (ARDS) is thought to be a typical pulmonary inflammation related to activation of polymorphonuclear leukocytes (PMNs) [1]. It is known that activated PMNs with phorbol myristate acetate (PMA) induced increases in filtration coefficient in isolated rat lungs [2]. It is also known that PMNs are activated during cardiopulmonary bypass [3] and therefore they may play a role in the pulmonary injury associated with this procedure [4]. Blood contact with synthetic surfaces during cardiopulmonary bypass causes a diffuse inflammatory reaction that includes neutrophil activation [5]. We postulated that during bypass, mechanical shear stress or contact with synthetic surfaces, such as membrane oxygenator, stimulates PMNs to increase the availability of adhesion molecules on their surfaces. Consistent with this notion, mechanical agitation for 30 seconds on a tube mixer induced an increase in the proportion of PMNs bearing receptors for C3b (C3b-R) [6]—now classified as CD11b/CD18 (Mac 1)—thus enabling them to adhere more readily to vascular endothelial cells. Once bound, PMNs may injure endothelial cells and increase pulmonary vascular resistance by releasing chemical mediators such as leukotrienes, oxygen radicals, and elastase.
Alternatively, or in addition, the endothelial cell injury caused by activated PMNs may result from an interaction between PMN adhesion molecules and an endothelial cell signaling pathway mediating conversion of xanthine dehydrogenase (XD) to xanthine oxidase (XO); in heart and lung, XO is present exclusively in the endothelial cells lining the associated vasculature [7]. Increased levels of endothelial XO would elevate intracellular superoxide anions (O2-) whose cytotoxicity would be expected to result in endothelial cell damage and increased pulmonary vascular permeability and resistance. If this is the case, and the primary sites for this inflammatory process are the endothelial cells, the effect should be inhibited by superoxide dismutase (SOD), a scavenger of superoxide radicals, and by allopurinol, an XO inhibitor. In this study, we addressed the question of whether stimulated PMNs increase pulmonary vascular permeability or resistance and, if so, whether reactive oxygen metabolites mediate the increase. To answer these questions, we performed experiments on isolated rat lungs.
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Material and methods
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Isolation of polymorphonuclear leukocytes
Human PMNs were isolated from heparinized blood obtained from a healthy adult who had no infectious foci and was taking no antiinflammatory medications. Blood samples were layered over Polymorphprep (Nycomed Pharmaceuticals, Oslo, Norway) and centrifuged at 500g for 35 minutes at room temperature, and the band of PMNs was harvested using a Pasteur pipette. The PMN fraction was resuspended in modified Hanks’ balanced salt solution (HBSS; Sigma, St. Louis, MO) without calcium, magnesium, or bicarbonate, and again centrifuged at 400g for 10 minutes. The cells were finally resuspended in HBSS to restore normal osmolarity and then counted using an automated cell counter (Coulter T-890, Coulter Electronics, Inc, Tokyo, Japan).
Immunofluorescence
The presence of adhesion molecules on the surface of the isolated PMNs was analyzed using an immunofluorescence technique. Stimulated and unstimulated PMNs (~106) were incubated with 2.9 µg of anti-CD18 monoclonal antibodies (MoAb; YFC118.3) for 20 minutes on ice. The immunofluorescence emitted from the PMNs was then analyzed using a flowcytometer on an Ortho Cytron Absolute (Ortho Diagnostic Systems, Raritan, NJ).
Isolated rat lung preparation
Thirty adult male Sprague-Dawley rats (252.6 ± 72.3 g) were used in this study. All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. After anesthetizing each animal with pentobarbital sodium (50 mg/kg IP), a tracheotomy was performed, and a 15-gauge luer stub adapter was inserted into the trachea (Clay Adams, Parsippany, NJ). The carotid artery was then catheterized (PE 50, Clay Adams), 500 units/kg of heparin was injected, and the rats were exsanguinated. At that point, we made a sternum-splitting incision and opened the pericardium. The pulmonary artery was catheterized by way of the right ventricle using polyethylene tubing (PE 200, Clay Adams) connected to silastic tubing (outer diameter 4.65 mm, inner diameter 3.35 mm; Dow-Corning, Midland, MI). The aorta was then ligated, and the left ventricle was catheterized. We flushed the lungs with 50 mL of saline until the effluent became clear and the lungs turned white. The venae cavae were then ligated, and the heart and lungs were removed en bloc.
After recording the weight of the preparation, including the heart, lungs, and catheters, we placed it in a Plexiglas box (B-345, Lustro Ware, Tokyo, Japan) and suspended it from a counterbalancing bar attached to a force displacement strain gauge transducer (FT pick up TB-611T, Nihon Kohden, Tokyo, Japan) (Fig 1). The trachea was connected to a compressed-air source so that the airway pressure could be maintained continuously at 2.0 cm H2O. The catheters into the pulmonary artery and the left ventricle were connected to arterial and venous reservoirs, respectively. The reservoirs were immersed in a water bath and warmed to 37°C, saturated with mixed gas containing 30% O2, 5% CO2, and 65% N2, and could be individually set at various heights to yield desired vascular pressures.
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Fig 1. Experimental setup for measuring pulmonary vascular permeability in isolated rat lungs. Rat lungs were placed in a Plexiglas box and perfused in an isogravimetric state under zone 3 conditions (pulmonary arterial pressure > pulmonary venous pressure > alveolar pressure). The perfusate was Krebs-Henseleit solution containing 6% bovine serum albumin. Weight gain was monitored continuously using a force transducer. Airway pressure (Paw), pulmonary arterial and venous pressures (Ppa and Ppv, respectively), and perfusate flow (Qp) were monitored continuously. Arterial and venous reservoirs were set at various heights to provide the desired vascular pressures.
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For this study, the lungs were kept in an isogravimetric state under zone 3 conditions (pulmonary arterial pressure > pulmonary venous pressure > alveolar pressure). The heart and lungs were perfused with Krebs-Henseleit buffer containing 6% bovine serum albumin (fraction V, Sigma), and a constant pressure-flow system was used throughout the experiments. Pulmonary venous (Pv) and pulmonary airway (Paw) pressures were adjusted to 2.5 and 2.0 cm H2O, respectively, and pulmonary arterial pressure (Ppa) was adjusted so that the lungs were neither gaining nor losing weight.
Experimental protocol
The objective of these experimental protocols was to determine whether activated PMNs increase pulmonary vascular permeability and resistance and, if so, whether superoxide anions produced by XO mediate the increases. To accomplish this, five series of experiments were carried out in five groups of isolated rat lungs. In the first series of experiments (unstimulated group; n = 6), 100 µL of 0.1 N NaOH, (vehicle for allopurinol) was injected into the pulmonary artery; ~30 minutes later, unstimulated PMNs (25 cells/µL) were injected. The protocol for the second series of experiments was identical to the first except that stimulated PMNs were used (stimulated group; n = 6). In the third series (Ab group; n = 6), we injected saline followed by stimulated PMNs (25 cells/µL) that had been incubated previously with CD18 MoAb. The fourth series of experiments (SOD group; n = 6) entailed injection of SOD (recombinant human Cu-Zn SOD; Wako Chemicals, Tokyo, Japan) at a final concentration of 80 units/mL followed by injection of stimulated PMNs. Finally, in the fifth series (ALLO group; n = 6), we injected 100 µL of 100 µM allopurinol (Sigma), an inhibitor of XO, followed by injection of stimulated PMNs. Stimulation of PMNs was achieved by shaking them gently in a glass vial for 10 seconds at room temperature (20°C ± 2°C). In the Ab group, stimulated PMNs were incubated with 2.9 µg of anti-CD18 MoAb (YFC118.3) per 106 PMNs for 20 minutes on ice. The PMN specimens were made up of 82.3% ± 7.3% neutrophils; the remaining cells were monocytes.
Before and after each injection of vehicle and again 90 minutes after injection of the PMNs (25 cells/µL of perfusate), we measured the filtration coefficient (K), an indicator of vascular permeability; to monitor the pulmonary hemodynamics, perfusate flow rate (Qp), Ppa, and pulmonary double occlusion (Pdo) pressure, which represents pulmonary capillary pressure, were measured [8]. After these measurements were obtained, either unstimulated or stimulated PMNs were injected into the pulmonary artery at the final concentration of 250 cells/µL of perfusate. The rat lungs were perfused with PMNs for ~10 minutes and then flushed with 50 mL of saline to washout intravascular PMNs that had not adhered to endothelial cells. They were then stored in a deep freezer at -80°C until myeloperoxidase (MPO) measurements were made.
Measurements
Pulmonary arterial and venous pressures and airway pressure were measured continuously with pressure transducers (P23ID, Gould Inc, Santa Ana, CA), and the rate of perfusate flow (Qp) was monitored using electromagnetic flow meters (FF050 and MF-27, Nihon Kohden), and they were recorded continuously on a polygraph (WT-687G, Nihon Kohden).
Calculation of the filtration coefficient
The filtration coefficient (K) was calculated as described previously [9]. Briefly, arterial and venous pressures were simultaneously increased by 3 cm H2O and the time-dependent increase in lung weight was measured. The gain in lung weight occurred in two phases; the standard interpretation of the weight gain curve is that there is an early gain due to increased vascular volume and slower gain resulting from continuous filtration. The increased vascular volume was completed within less than 3 minutes; consequently, this component could be discriminated from the filtration component by plotting the log of the weight gain as a function of time [9, 10]. By using the method of least squares to fit a line to the later phase (data obtained from the last 7 minutes) and extrapolating back to time zero, we obtained the initial filtration rate; K was calculated by dividing the initial filtration rate by the applied microvascular pressure increment and normalizing to 1 g of wet lung weight.
Myeloperoxidase assay
As an index of PMN accumulation in the lungs, we measured MPO using a luminescence technique. The frozen rat lungs were thawed, a volume of 0.02% cethyltrimethyl ammonium bromide (CTAB; Wako Chemicals) equivalent to 5 times the lung weight was added, and the lungs were homogenized at 0°C. The homogenates were centrifuged at 10,000 rpm and 4°C for 15 minutes in a high-speed, refrigerated centrifuge (Hitachi 18PR-3, Hitachi, Tokyo, Japan). The pellets were frozen rapidly in liquid nitrogen, placed on ice, and then homogenized by sonication (UD201, Tomy Seiki, Tokyo, Japan) for 30 seconds at 30 W (30 times of 0.5 second burst plus 0.5 second stand). Freezing and sonication of the pellets were repeated 7 times. The homogenates were centrifuged at 12,000 rpm and 4°C for 15 minutes, and the supernatants were collected for assay. Initially, standard reagent (50 µM SOD, 20 µL; 2 mM desferrioxamine, 20 µL; 50 mM Kbr, 20 µL; 40 mM H2O2, 25 µL; 0.2 M acetate buffer, 1 mL; distilled water, 765 µL; and 0.2 mM 2-methyl-6-[p-methoxyphenyl]-3, 7-dihydroimidazo[1,2-a] pyrazin-3-one [MCLA; Tokyo Kasei Organic Chemicals, Tokyo, Japan], 100 µL) was injected into a luminescence reader (BLR301, Aloca, Tokyo, Japan), and the basal luminescence was characterized for 3 minutes. The luminescences of the test specimens (50 µL of supernatant) were then assayed.
Statistical analysis
Data are expressed as means ± SD and were analyzed by analysis of variance (ANOVA). Values of p less than 0.05 were considered significant.
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Results
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Immunofluorescence
Comparison of stimulated and unstimulated PMNs by analysis of their immunofluorescence revealed that CD18 and CD11b were upregulated on the surfaces of stimulated PMNs (Fig 2, left panels). In contrast, CD11a and CD11c were unaffected by mechanical stimulation (Fig 2, right panels).
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Fig 2. Histograms depicting immunofluorescence reflecting expression of the ß2 integrins CD18, CD11a, CD11b, and CD11c on the surface of the unstimulated polymorphonuclear leukocytes (PMNs) (hatched histograms) and PMNs stimulated by being shaken in a glass vial (closed histograms); the open histograms represent negative controls.
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Pulmonary vascular permeability
Addition of vehicle or unstimulated PMNs had no significant effect on baseline values for the K, which was measured before and after injection of vehicle and after injection of PMNs (4.97 ± 1.14, 4.45 ± 0.87, 5.45 ± 3.23 mg · cm H2O-1 · min-1 · g-1, respectively). On the other hand, after stimulated PMNs were injected, there was a fivefold increase (p < 0.05 vs unstimulated group) in the K (5.63 ± 1.61, 5.03 ± 1.17, 26.64 ± 9.19 mg · cm H2O-1 · min-1 · g-1, respectively), suggesting that significant injury to the endothelium had occurred (Fig 3). In the Ab group, the increase in the K induced by stimulated PMNs was blocked completely by preincubation with antibodies against CD18 (5.92 ± 0.97, 5.41 ± 2.21, 5.57 ± 2.32 mg · cm H2O-1 · min-1 · g-1, respectively) (p < 0.05 vs stimulated group). This increase was also inhibited by SOD (5.26 ± 1.13, 4.85 ± 0.84, 5.32 ± 0.51 mg · cm H2O-1 · min-1 · g-1, respectively) (p < 0.05 vs stimulated group) and by allopurinol (5.13 ± 1.51, 5.03 ± 1.61, 5.74 ± 2.29 mg · cm H2O-1 · min-1 · g-1, respectively) (p < 0.05 vs stimulated group). Thus, blockade of CD18 adhesion molecules, scavenging of superoxide anion, or blockade of XO appears to be protective against stimulated PMN-induced endothelial cell damage.
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Fig 3. Effects of anti-CD18 monoclonal antibody, superoxide dismutase (SOD), and the xanthine oxidase inhibitor allopurinol on the increase in pulmonary vascular permeability (filtration coefficient) elicited by stimulated polymorphonuclear leukocytes (PMNs). Values are means ± SD. *p < 0.05 vs stimulated PMN group.
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Pulmonary vascular resistance
For all five groups of rats, pulmonary hemodynamics were not affected significantly by any of the treatment protocols (Table 1).
Myeloperoxidase assay
The MPO levels measured in the stimulated group (57.8 ± 27.4 kcpm) were significantly higher than those in the unstimulated group (5.86 ± 6.73 kcpm) (p < 0.05) (Fig 4). This suggests that stimulated PMNs accumulated in the lungs to significantly greater extent than unstimulated cells. The increased MPO seen with stimulated PMNs was blocked completely by preincubation with anti-CD18 MoAb (5.10 ± 5.06 kcpm; Ab group) (p < 0.05), but was unaffected by exposing cells to SOD (77.93 ± 20.82 kcpm; SOD group). In the ALLO group, MPO levels were 28.04 ± 13.04 kcpm, which were significantly lower than those in the stimulated group (p < 0.05), but significantly higher than those in the unstimulated or Ab groups (p < 0.05).
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Fig 4. Myeloperoxidase (MPO) was measured in each experimental group. In lungs injected with stimulated polymorphonuclear leukocytes (PMNs), MPO levels were higher than in lungs injected with unstimulated PMNs or with stimulated PMNs incubated with anti-CD18 monoclonal antibody. On the other hand, superoxide dismutase (SOD) did not affect elevated MPO levels induced by stimulating PMNs. The MPO levels in the ALLO group were significantly lower than those in the stimulated group, but significantly higher than those in the unstimulated and antibody groups. Values are means ± SD. *, # p < 0.05 vs stimulated and unstimulated PMN groups, respectively.
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When the data were considered together, we found a significant association between the K and MPO levels in the unstimulated and stimulated groups (n = 12, r = 0.91, p < 0.0001) (Fig 5). No significant correlation was found among any of the other treatment groups (Fig 5).
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Fig 5. For each experimental group, pulmonary vascular filtration coefficients are plotted as a function of myeloperoxidase (MPO) level. Each point represents a single experiment (open circles, unstimulated polymorphonuclear leukocytes [PMNs]; closed circles, stimulated PMNs; closed triangles, anti-CD18 antibody; open squares, allopurinol; closed squares, superoxide dismutase). The regression line was obtained from the data of stimulated and unstimulated groups.
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Comment
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Little information about the increase in pulmonary vascular permeability caused by mechanically stimulated PMNs is currently available. In this study, we found that when injected into the pulmonary circulation, stimulated PMNs increase pulmonary vascular permeability by increasing the levels of superoxide metabolites within the endothelium. Consistent with this idea, CD11b/18 (Mac 1) adhesion proteins on the surface of PMNs were upregulated by mechanical stimulation, and increases in vascular permeability were blocked when stimulated PMNs were treated with anti-CD18 MoAb. We postulate, therefore, that stimulated PMNs injured vascular endothelial cells by attaching to their surfaces. Once attached, PMNs appear to stimulate production of superoxide metabolites by XO, an effect that was blocked by both SOD, a superoxide scavenger, and allopurinol, an XO antagonist.
Our analysis of cellular immunofluorescence showed that CD11b and CD18 were both upregulated on the surface of stimulated PMNs. CD11b/18 (Mac 1), a family of ß2 leukocyte integrins, is one of the principle glycoprotein-derived adhesion molecules that enable PMNs to attach firmly to endothelial cells. A previous report showed that agitation caused an increase in the proportion of granulocytes bearing receptors for C3b (C3b-R) and lymphocytes bearing receptors for Fc gamma-R [6]. C3b, which is also classified as Mac 1, uses ICAM-1 or ICAM-2 located on the surface of endothelial cells as counterligands.
Increasing the availability of CD18 on the surface of PMNs by agitation in a glass vial would be expected to cause an increase in adhesiveness of PMNs. When we injected stimulated PMNs into isolated rat lungs, the pulmonary vascular permeability was increased. This elevation in permeability, however, was blocked completely by preincubation of stimulated PMNs with anti-CD18 MoAb. In addition, MPO levels in the Ab group were no different from the levels in the unstimulated group and were, therefore, low compared with those of the stimulated PMNs group. The ability of anti-CD18 MoAb to block the effects of stimulated PMNs confirms the importance of PMN adhesion to endothelial cells by CD18. Both LFA-1 (lymphocyte function-associate Ag-1) and Mac 1 were assembled with subunits of ß2 integrins (CD11a and CD11b, respectively) and CD18 as a common subunit. Because anti-CD18 MoAb may block both LFA-1 and Mac 1, it could block completely the elevation in permeability.
The evidence that Cu-Zn SOD blocked the increase in permeability mediated by stimulated PMNs demonstrates that superoxide anion (O2-) participated in the increase in permeability. SOD transforms superoxide anion to hydrogen peroxide (H2O2), which is, in turn, transformed to water (H2O) by catalase contained in peroxisomes within the cytoplasm. In this way, living cells are protected from oxygen injury. Although some SOD is present in endothelial cells, it is primarily stored in erythrocytes. In that regard, an earlier report suggested that erythrocytes provide a protective effect against lung injury [11]. Our observations are consistent with this earlier finding because in the erythrocyte-free system used in the present study, SOD had to be administered exogenously to achieve a protective effect.
The results discussed so far indicate that superoxide anion plays a crucial role in mediating the observed increase in pulmonary vascular permeability. A key question then is how is the superoxide anion generated? Previous reports indicate that during respiratory bursts, NADPH oxidase in the plasma membranes of PMNs serves as an important source of superoxide anion [12], whereas XO is a major source of superoxide anion in cytoplasm of the endothelial cells [7]. Stresses such as ischemia-reperfusion [13], attachment of PMNs to the surface of endothelial cells [14], and hypoxia [15] may induce conversion of XD to XO. The metabolic pathway for the major purinergic compounds adenine and guanine entails converting them into xanthine, which is then oxidized to uric acid by XO; superoxide anion is generated in the process. Superoxide anion then undergoes conversion to hydroxyl radicals ( · OH) by the Fenton or Harber-Weiss reaction [16]. When there is sufficient SOD, superoxide anion is scavenged, and the reduction of stored Fe3+ into Fe2+ is attenuated [17]. In the presence of excess superoxide anion that could not be scavenged by SOD, stored Fe3+ is reduced to Fe2+, and hydrogen peroxide undergoes conversion to hydroxyl radicals by the Fenton reaction. As both PMNs and endothelial cells are potential sources of superoxide anion, SOD also may scavenge PMN-derived superoxide.
We observed that increases in the filtration coefficient induced by stimulated PMNs were blocked by allopurinol. This indicates that inhibition of XO decreased the generation of superoxide anion and presumably reduced damage to endothelial cells. However, when we analyzed MPO levels in each experimental group, the situation seems less straightforward. The MPO levels in the ALLO group were significantly lower than those in the stimulated group but significantly higher than those in both the unstimulated and Ab groups. This situation suggests that allopurinol may not only inhibit the enzyme activity of XO but also the binding of Mac 1 to ICAM-1. There are four possible mechanisms by which Mac 1 binding to ICAM-1 might be reduced: (1) downregulation of ICAM-1, (2) inhibition of upregulation of ICAM-1, (3) shedding of ICAM-1, and (4) direct inhibition of the interaction between Mac 1 and ICAM-1. It is unlikely that ICAM-1 was downregulated because as a member of an immunoglobulin superfamily, ICAM-1 would be expected to be continuously present on the surface of endothelial cells. Alternatively, allopurinol may accelerate shedding of ICAM-1, or directly inhibit the interaction of ICAM-1 and Mac 1, but there is presently no published evidence supporting these scenarios. Stimuli such as interleukin-1 (IL-1), tumor necrosis factor-
(TNF-), and lipopolysaccharides (LPS) are known to upregulate endothelial ICAM-1 [18], and then the ICAM-1 is shed as soluble ICAM-1 [18]. Many studies support the conclusion that ICAM-1 upregulation requires several hours as it usually requires gene transcription and protein synthesis [19]. There is another possible mechanism that may reduce MPO levels in the lungs. If substantial release of MPO had occurred within the vasculature as a result of allopurinol-induced enzyme secretion from neutrophils, measurement of MPO in lung tissue may be an underestimate of the lung content of neutrophils [20]. The fact that allopurinol blocked the increase in lung permeability suggests that superoxide anions generated by XO may play a role in this increase.
In conclusion, the present study shows that mechanically stimulated PMNs increase pulmonary vascular permeability and that superoxide anion, most likely generated by XO possibly within endothelial cells, mediates the increase.
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Accepted for publication July 13, 1999.
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Abstract
Introduction
