1 Institute for Environmental Medicine and 4 Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6068; 2 Departments of Anesthesiology and Pharmacology, University of Illinois at Chicago, Chicago, Illinois 60612; and 3 Department of Physiology and Biophysics, Louisiana State University Medical Center, Shreveport, Louisiana 39532
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ABSTRACT |
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The pulmonary endothelium is susceptible to oxidative insults. Catalase conjugated with monoclonal antibodies (MAbs) against endothelial surface antigens, angiotensin-converting enzyme (MAb 9B9) or intercellular adhesion molecule-1 (MAb 1A29), accumulates in the lungs after systemic injection in rats (V. Muzykantov, E. Atochina, H. Ischiropoulos, S. Danilov, and A. Fisher. Proc. Natl. Acad. Sci. USA 93: 5213-5218, 1996). The present study characterizes the augmentation of antioxidant defense by these antibody-catalase conjugates in isolated rat lungs perfused for 1 h with catalase conjugated with either MAb 9B9, MAb 1A29, or control mouse IgG. Approximately 20% of the injected dose of Ab-125I-catalase accumulated in the perfused rat lungs (vs. <5% for IgG-125I-catalase). After elimination of nonbound material, the lungs were perfused further for 1 h with 5 mM hydrogen peroxide (H2O2). H2O2 induced an elevation in tracheal and pulmonary arterial pressures (126 ± 7 and 132 ± 5%, respectively, of the control level), lung wet-to-dry weight ratio (7.1 ± 0.4 vs. 6.0 ± 0.01 in the control lungs), and ACE release into the perfusate (436 ± 20 vs. 75 ± 7 mU in the control perfusates). Both MAb 9B9-catalase and MAb 1A29-catalase significantly attenuated the H2O2-induced elevation in 1) angiotensin-converting enzyme release to the perfusate (215 ± 14 and 217 ± 38 mU, respectively), 2) lung wet-to-dry ratio (6.25 ± 0.1 and 6.3 ± 0.3, respectively), 3) tracheal pressure (94 ± 4 and 101 ± 4%, respectively, of the control level), and 4) pulmonary arterial pressure (103 ± 3 and 104 ± 7%, respectively, of the control level). Nonconjugated catalase, nonconjugated antibodies, nonspecific IgG, and IgG-catalase conjugate had no protective effect, thus confirming the specificity of the effect of MAb-catalase. These results support a strategy of catalase immunotargeting for protection against pulmonary oxidative injury.
endothelium; hydrogen peroxide; angiotensin-converting enzyme; intercellular adhesion molecule-1
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INTRODUCTION |
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THE PULMONARY VASCULAR ENDOTHELIUM is anatomically and physiologically predisposed to oxidative insults. Inhalation of oxygen or ozone, as well as exposure to a hypoxic environment, results in the generation of oxidants in the lung (17, 29). Lung ischemia stimulates generation of oxidants by the pulmonary endothelial cells and thus leads to autoxidative damage (15). Activated leukocytes that accumulate in the pulmonary vasculature release oxidants that can damage the endothelium (7, 27). Therefore, the pulmonary endothelium is susceptible to damage by oxidants generated either intracellularly or extracellularly. Oxidative injury to the pulmonary endothelium leads to serious pathological consequences including, but not limited to, 1) overexpression of surface adhesion molecules (30, 44, 49) and secondary leukocyte-mediated lung injury (7, 27, 29), 2) fibrin deposition in the pulmonary vasculature and lung fibrosis (12), and 3) disregulation of vascular tone and pulmonary hypertension (33). Therefore, protection of the pulmonary endothelium against oxidative injury represents an important strategy for therapeutic intervention in different lung diseases, including adult respiratory distress syndrome (29).
Two antioxidant enzymes, superoxide dismutase [SOD; that dismutes superoxide anion into hydrogen peroxide (H2O2)] and catalase (that degrades H2O2 into water) have been extensively studied during the last two decades for their potential effectiveness in antioxidative therapy (21, 29, 31). Both catalase and SOD, however, undergo rapid elimination from the bloodstream and demonstrate poor intracellular delivery (21). Chemical modification or encapsulation of catalase and SOD into liposomes prolongs their lifetime and facilitates cellular uptake of the enzymes (4, 18) and thus enhances their antioxidative potential. For example, intratracheal administration of liposomal formulations of antioxidant enzymes protects lung tissue against oxidative injury in laboratory animals (3). Pulmonary endothelial cells, however, are not readily accessible from the alveolar space. Intravascular administration seems to be a more appropriate route for drug delivery to these cells. To effectively utilize this route of administration, however, a drug must possess an affinity for endothelial cells. Such an affinity could provide cell-specific targeting and prolonged association of a drug with the endothelium. Based on this consideration, we proposed and explored the conjugation of antioxidant enzymes with antibodies that recognize surface endothelial antigens, angiotensin-converting enzyme (ACE) and intercellular adhesion molecule-1 (ICAM-1).
ACE is a membrane glycoprotein that is constitutively expressed on the surface of the vascular endothelium (13). A major portion of the ACE molecule is exposed to the bloodstream from the luminal surface of endothelial cells. Importantly, ACE content in the pulmonary vasculature is markedly higher than that in the systemic vasculature (16). In accordance with this fact, previous results documented that anti-ACE monoclonal antibody (MAb) 9B9 accumulates selectively in the lungs of various animal species, including humans, after systemic injection (9, 39) and that catalase conjugated with MAb 9B9 accumulates in rat lungs after systemic injection (37). ICAM-1 is also a glycoprotein that is constitutively expressed on the luminal surface of endothelial cells (1, 46). Previous reports from several laboratories (35, 42, 51) have documented that MAbs against ICAM-1 accumulate in the lung after systemic administration. Importantly, inflammatory mediators (e.g., tumor necrosis factor, oxidants, and C5a) upregulate the surface expression of ICAM-1 in endothelial cells (1, 6, 34, 35, 46, 49). In accordance with this fact, pulmonary uptake of radiolabeled anti-ICAM-1 MAb 1A29 is enhanced in rats challenged with these proinflammatory mediators compared with control rats (34, 35, 43).
Previous results that support the feasibility of immunotargeting of antioxidant enzymes to the pulmonary endothelium are based on the distribution of radiolabeled catalase conjugated with a carrier antibody after intravascular injection in rats (37). Although this is important quantitative information concerning the magnitude and kinetics of accumulation of the antibody-conjugated enzyme in the lungs, it does not provide any insight into the activity of the conjugates accumulated in the lung and the magnitude of antioxidant lung defense augmentation. To obtain such information, we undertook a series of experiments that were designed to address the effects of antibody-conjugated catalase on the pulmonary vascular injury induced by H2O2 in the isolated rat lung. Perfusion of isolated lungs under conditions of constant ventilation and perfusion rate allows for an assessment of the effects of a defined oxidant on the pulmonary vasculature in the whole organ without the potentially confusing influence of blood and systemic effects. ACE release from the pulmonary vasculature into the perfusate solution was used as a cell-specific index of endothelial injury. The results show that catalase immunotargeting to endothelial antigens affords significant attenuation of the endothelial damage in our model of acute oxidative injury and thus may be further explored in in vivo models.
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MATERIALS AND METHODS |
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Reagents. The following materials were used in the study: Iodogen from Pierce (Rockford, IL); Na125I from Amersham (Arlington Heights, IL); fatty acid-free BSA from Boehringer Mannheim (Indianapolis, IN); trichloroacetic acid, dimethylformamide, H2O2, mouse IgG, and components of buffer solutions from Sigma (St. Louis, MO); o-phthalaldehyde and Z-Phe-His-Leu from Serva (Heidelberg, Germany); lyophilized bovine liver catalase (activity 20,000 U/mg) from Fluka (Ronkonkoma, NY); Sephacryl S-200HR from Pharmacia-LKB; streptavidin (SA) and 6-biotinylaminocaproic acid N-hydroxysuccinimide ester [biotin-X-NHS (BxNHS)] from Calbiochem (San Diego, CA); and avidin-biotin complex kit for immunoperoxidase staining (biotinylated horse antibodies against mouse IgG and SA-peroxidase complex) from Vector Laboratories (Burlingame, CA). Protein concentration was determined by a Bio-Rad protein microassay kit (Hercules, CA). MAbs against ACE (MAb 9B9, mouse IgG1 class) and ICAM-1 (MAb 1A29, mouse IgG1 class) have been characterized previously (9, 42).
Biotinylation and radiolabeling of proteins. Control mouse IgG, anti-ACE MAb 9B9, and anti-ICAM-1 MAb 1A29 were biotinylated at a 10-fold molar excess of the biotinylating reagent BxNHS as described previously (40). Catalase was biotinylated by the same reagent at a 15-fold molar excess as previously described (39). Briefly, a fresh 0.1 M solution of BxNHS in anhydrous dimethylformamide was added to the solution of a protein (1 mg/ml) in borate-buffered saline (pH 8.0). After 1 h of incubation on ice, an excess of nonreacted BxNHS was eliminated by overnight dialysis. Previous studies documented that biotinylation under the described conditions provides covalent coupling of 1-2 SA-accessible biotin residues/protein molecule but does not lead to a detectable reduction in the antigen-binding activity of antibodies or the enzymatic activity of catalase (36, 37, 40). In the following text, the biotinylated proteins are designated b-IgG, b-MAb, and b-catalase. Proteins (b-catalase, IgG, or antibodies) were radiolabeled with 125-I with Iodogen-coated tubes according to the manufacturer's recommendation.
Preparation of b-catalase-SA-b-IgG complexes. To construct trimolecular heteropolymer complex b-catalase-SA-b-IgG, we used a two-step procedure developed in our laboratory and previously described (36, 37). Briefly, at the first step, SA and b-catalase were mixed at a molar ratio of SA to b-catalase of 10 and incubated for 1 h on ice to form a bimolecular complex, b-catalase-SA. An excess of nonbound SA was eliminated by Sephacryl S-200HR gel filtration as previously described (36). At the second step, the bimolecular complex b-catalase-SA was incubated with b-MAb 9B9, b-MAb 1A29, or b-IgG to form a trimolecular complex b-catalase-SA-b-antibody. Antibody-conjugated catalase retains its enzymatic activity, binds to immobilized antigen in vitro, and degrades H2O2 in the antigen-coated wells (36).
Perfusion of the isolated rat lung. Sprague-Dawley male rats weighing 170-200 g were anesthetized with pentobarbital sodium (50 mg/kg ip) and prepared for isolated lung perfusion with recirculating perfusate as previously described (38). The trachea was cannulated, and the lungs were ventilated with a humidified gas mixture (Airco, Philadelphia, PA) containing 5% CO2 and 95% air. Ventilation was performed with an SAR-830 rodent ventilator (CWE, Ardmore, PA) at 60 cycles/min, 2-ml tidal volume, and 2-cmH2O end-expiratory pressure. The thorax was then opened, and a cannula was placed in the main pulmonary artery through the transected right ventricle. The left atrium was transected to allow free outflow of the perfusate. The lungs were isolated from the thorax and initially perfused in a nonrecirculating manner for a 5-min equilibration period to eliminate blood from the pulmonary vascular bed. Then lungs were transferred to a water-jacketed perfusion chamber maintained at 37°C. Perfusion through the pulmonary artery was maintained by a peristaltic pump at a constant flow rate of 10 ml/min. Perfusate flowing from the transected atrium was collected in the perfusion chamber and recirculated. The perfusate (45 ml/lung) was Krebs-Ringer buffer (KRB; pH 7.4) containing 10 mM glucose and 3% fatty acid-free BSA. The perfusate was filtered through a 0.4-µm filter before perfusion to eliminate particulates. Intratracheal and pulmonary arterial pressures were continuously recorded throughout the experiment with pressure transducers PM 131TC and P23DC (Statham Instruments, Oxnard, CA), direct-writing oscillographs (Gould, Cleveland, OH), and alternate current recorders (Primeline, Sun Valley, CA). Zero reference for perfusion pressure was determined at the end of each experiment and was defined as the pressure measured at the experimental flow rate without the lungs being connected to the circuit.
Experimental protocols. After isolation of the lungs as described in Perfusion of the isolated rat lung, the lungs were initially perfused with the KRB-BSA solution for a 5-min equilibration period. Then 1 µg of radiolabeled conjugate (anti-ACE-125I-catalase, anti-ICAM-1-125I-catalase, or IgG-125I-catalase) was added to the perfusion. After a 1-h perfusion, the lungs were perfused in a nonrecirculating manner for 5 min with the KRB-BSA solution to eliminate nonbound radiolabeled material. The lungs were removed from the chamber, rinsed with saline, and blotted with filter paper; and the extraneous cardiac and bronchial structures were dissected away. The left lobe was removed and blotted with filter paper, the wet weight was determined, and the radioactivity was measured in a gamma counter and is expressed as a percentage of perfused radioactivity (injected dose) per gram of lung tissue (%ID/g).
In the second protocol, after an initial 5-min stabilization period, isolated rat lungs were perfused for 1 h under standard conditions with perfusate containing 30 µg of catalase conjugated with mouse IgG (IgG-Cat), anti-ACE MAb 9B9 (9B9-Cat), or anti-ICAM MAb 1A29 (1A29-Cat). Perfusate for the control lungs was conjugate-free KRB-BSA solution. After 1 h of perfusion, nonbound conjugates were eliminated by nonrecirculating perfusion of conjugate-free KRB-BSA solution for 5 min, and the lungs were again perfused in a recirculating manner for 1 h with a KRB-BSA solution containing 2 or 5 mM H2O2. After perfusion, the lung lobes were processed by the procedure described above to determine the wet-to-dry weight ratio. Aliquots of the perfusate (0.1 ml) were collected after every 15 min of perfusion and frozen in liquid nitrogen.
Determination of ACE activity in the perfusate. ACE activity in the perfusate was measured with a fluorometric assay as the rate of generation of His-Leu formed from the ACE substrate Z-Phe-His-Leu (19). Ten microliters of the perfusate were added to 200 µl of 50 mM Tris · HCl-0.15 M NaCl buffer, pH 8.3, containing 0.5 mM substrate. Samples of the perfusate were incubated at 37°C for 120 min, and then the reaction was terminated by the addition of 1.5 ml of 0.28 N NaOH. o-Phthalaldehyde (1 mg in 100 µl of methanol) was added for 10 min before this reaction was stopped with 200 µl of 2 N HCl. His-Leu was measured with a fluorescence spectrophotometer at an excitation wavelength of 363 nm and an emission wavelength of 500 nm. The results were calculated as milliunits of ACE activity per total perfusate (45 ml), where 1 mU represents the generation of 1 nmol His-Leu/min.
Separation of the perfusate into aqueous and detergent phases was performed according to the procedure for the separation of integral and surface membrane proteins developed by Bordier (5). Briefly, an aliquot of the perfusate was prepared in 200 µl of 10 mM Tris · HCl, pH 7.4, 150 mM NaCl, and 1% Triton X-114 at 0°C. The clear sample was overlaid on 300 µl of a 6% (wt/vol) sucrose cushion in 10 mM Tris · HCl, pH 7.4, 150 mM NaCl, and 0.06% Triton X-114; incubated for 5 min at 30°C for condensation; and centrifuged for 5 min at 300 g in a swinging-bucket rotor. The supernatant was collected, and fresh ice-cold Triton X-114 was added to a final concentration of 0.5%. After dissolution of the mixture at 0°C, the sample was again overlaid on the sucrose cushion previously used, incubated for 5 min at 30°C, and centrifuged on the previous detergent phase. At the end of separation, ACE activity was assessed in the aqueous (upper layer) and detergent phases as described above.
Immunoperoxidase staining of ACE and ICAM-1 in the rat lungs. Binding of anti-ACE MAb 9B9 and anti-ICAM-1 MAb 1A29 to their epitopes in rat lung tissue was visualized by secondary biotinylated antibody and SA conjugated with peroxidase. Briefly, rat lungs were perfused for 10 min with the KRB-BSA solution and then fixed by an intratracheal infusion of 4% paraformaldehyde-sodium cacodylate buffer. The fixed lungs were frozen in liquid nitrogen, cut into 8-µm sections, and preincubated with nonimmune rat serum. Endogeneous peroxidase activity in the lung tissue was blocked by incubation with 0.3% H2O2 for 10 min. The sections were incubated for 1 h with MAb 9B9 or MAb 1A29 diluted in PBS-1% BSA (final concentration 10 µg/ml). For a negative control, primary antibodies were replaced by nonspecific mouse IgG. After being washed, sections were incubated with the biotinylated goat antibody against mouse IgG followed by washing and incubation with an avidin-biotinylated horseradish peroxidase complex and developed with a 3,3'-diaminobenzidine-stabilized solution. The sections were counterstained with hematoxylin and mounted with Mowiol.
Statistical analysis. Unless specifically indicated, data are expressed as means ± SE. Comparisons were made with one-way ANOVA followed by the Student-Newman-Keuls method. The level of significance was taken as P < 0.05.
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RESULTS |
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Interaction of carrier antibodies and antibody-conjugated catalase with the pulmonary vasculature in perfused rat lungs. First, we visualized the binding of nonmodified anti-ACE MAb 9B9 and anti-ICAM-1 MAb 1A29 to their epitopes in frozen tissue sections of normal rat lungs. Figure 1 shows staining of the rat lung tissue with these antibodies with an indirect immunoperoxidase technique. Both antibodies display abundant binding to the pulmonary tissue. Mouse IgG, used in the present study as a negative control, produced no detectable staining in the rat lung tissue.
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Next, we determined the accessibility of pulmonary ACE and ICAM-1 for circulating ACE and ICAM-1 MAbs. Isolated rat lungs were perfused for 1 h with radiolabeled IgG, MAb 9B9, or MAb 1A29. After perfusion, nonbound radioactivity was eliminated by a single-pass perfusion of antibody-free perfusate for 5 min. Radiolabeled MAb 9B9 and MAb 1A29 accumulated specifically in the perfused rat lungs (Fig. 2A). Pulmonary uptake of 125I-MAb 9B9 and 125I-MAb 1A29 was 24.6 ± 2 and 21 ± 1%ID/g, respectively. In a similar experiment, we assessed the pulmonary uptake of 125I-catalase conjugated with IgG, MAb 9B9, or MAb 1A29. Nonconjugated 125I-catalase did not accumulate in the lung after 1 h of perfusion (<1%ID/g). In contrast, perfusion of the rat lungs with 125I-catalase conjugated with MAb 9B9 and MAb 1A29 led to uptakes of 18.6 ± 0.4 and 21.1 ± 1.7%ID/g, respectively (Fig. 2B). Therefore, catalase conjugated to either anti-ACE or anti-ICAM-1 antibody binds to vascular ACE or ICAM-1 as effectively as to the respective nonmodified carrier antibodies. IgG-Cat displayed four times lower uptake in the lung.
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In a separate experiment, we perfused normal rat lungs for 1 h with 100 µg of either MAb 9B9, MAb 1A29, or control IgG to assess the possible effects of these antibodies on the physiological and biochemical parameters of pulmonary oxidative injury. Neither antibody induced detectable alterations in tracheal or pulmonary arterial pressure, lung wet-to-dry weight ratio, or ACE activity in the perfusate.
Oxidative injury to the isolated rat lungs induced by perfused H2O2. Tracheal and mean pulmonary arterial pressures were continuously recorded during the experiment in all groups and were constant during the entire duration of control perfusion without H2O2. A low dose of H2O2 (0.2 mM) induced no detectable alterations in these physiological lung parameters. Perfusion of rat lungs with 5 mM H2O2 led to an elevation in peak tracheal pressure to 126 ± 7% of the control level at the end of the perfusion period. This concentration of H2O2 also induced a rapid but transient elevation in pulmonary arterial pressure that was observed ~5 min after the addition of H2O2 and declined a few minutes thereafter. About 30-40 min after the addition of 5 mM H2O2, the pulmonary arterial pressure again increased gradually but progressively and attained a value of 132 ± 5% of baseline at the end of perfusion. Perfusion with 10 mM H2O2 led to obvious generalized edema in >50% of perfused lungs, with a dramatic elevation in both peak tracheal pressure and pulmonary arterial pressure as early as 10-15 min after the infusion of H2O2. About 30-40% of lungs perfused with 10 mM H2O2 developed such a rapid and profound edema, and these lungs were not analyzed further.
Figure 3A shows the changes in wet-to-dry weight ratio in rat lungs perfused with 0.2-10 mM H2O2. With a low dose (0.2 mM), there was no significant elevation in the lung wet-to-dry weight ratio. Perfusion with higher concentrations of H2O2 led to significant elevations in the lung wet-to-dry weight ratio, with a near doubling of this parameter after perfusion of 10 mM H2O2.
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Antibody-conjugated catalase abrogates H2O2-induced elevation in the vascular permeability, tracheal pressure, and pulmonary arterial pressure. For immunotargeting of catalase, the lungs were perfused for 1 h with 30 µg of catalase conjugated with MAb 9B9, MAb 1A29, or control IgG. After 5 min of nonrecirculating perfusion with conjugate-free perfusate, the lungs were perfused for 1 h with 5 mM H2O2. In a control experiment, we assessed the effect of nonconjugated IgG, MAb 1A29, and catalase on the H2O2-induced injury in the perfused rat lung. Perfusion of these proteins (100 µg/lung) had no effect on the elevation in lung wet-to-dry weight ratio and ACE activity in the perfusate caused by subsequent perfusion of 5 mM H2O2 (data not shown). The control conjugate (IgG-Cat) did not attenuate the H2O2-induced elevation in the lung wet-to-dry weight ratio (Fig. 5). In contrast, perfusion of 9B9-Cat or 1A29-Cat provided reproducible and significant attenuation of the H2O2-induced elevation in the lung wet-to-dry weight ratio (6.25 ± 0.1 and 6.3 ± 0.3, respectively, vs. 7.07 ± 0.4; P < 0.05). Therefore, the lung wet-to-dry weight ratio of the lungs treated with 9B9-Cat or 1A29-Cat and perfused with 5 mM H2O2 did not differ significantly from that of lungs perfused with H2O2-free perfusate (6.0 ± 0.01). In a similar experiment, 9B9-Cat provided complete protection against 2 mM H2O2: wet-to-dry lung weight ratio was elevated to 6.7 ± 0.1 after perfusion of H2O2 (P < 0.05 compared with the control group) but was equal to 6.0 ± 0.1 after perfusion of H2O2 in the lungs pretreated with 9B9-Cat (P < 0.05 compared with the H2O2 group). IgG-Cat had no protective effect against 2 mM H2O2 (wet-to-dry weight ratio 7.1 ± 0.8; P < 0.05 compared with the control group).
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Antibody-conjugated catalase attenuates H2O2-induced ACE release. Figure 7 shows that 9B9-Cat and 1A29-Cat significantly attenuated the H2O2-induced elevation in ACE activity in the perfusate to the levels of 215 ± 14 and 217 ± 38 mU, respectively, vs. 436 ± 20 mU after perfusion with 5 mM H2O2; the differences are significant at P < 0.005. Perfusion with IgG-Cat had no protective effect but rather aggravated the H2O2-induced injury (566 ± 105 mU). Thus, under the described conditions, neither conjugate afforded complete abrogation of the H2O2-induced elevation in ACE activity in the perfusate (control level 75 ± 7 mU). Both immune conjugates, nevertheless, provided a >60% reduction of this parameter of oxidative endothelial injury. Perfusion of 9B9-Cat also provided significant protection against the elevation in perfusate ACE activity induced by 2 mM H2O2 (195 ± 21 vs. 311 ± 39 mU; P < 0.05; n = 4 lungs for both groups). In contrast, IgG-Cat provided no significant protection against 2 mM H2O2 (265 ± 15 mU; n = 3 lungs).
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DISCUSSION |
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Studies in cell culture, perfused organs, and animal models published during the last two decades provide a body of evidence that oxidants (e.g., superoxide anion, H2O2, hydroxyl radical, peroxynitrite) play an important role in the initiation, augmentation, and potentiation of endothelial injury in inflammation, ischemia-reperfusion, hypoxia, hyperoxia, and other pathological conditions (7, 20, 21, 29, 31). Several groups (3, 4, 18, 21, 31) have attempted to utilize the antioxidant enzymes SOD and catalase, as well as various derivatives of these enzymes, to protect various cells against oxidative injury. The present study represents a new step in the development of a novel strategy for the targeted delivery of protective antioxidant enzyme to the pulmonary endothelium.
Muzykantov et al. (41) postulated a decade ago that an antioxidant enzyme conjugated with a specific endothelial ligand will bind to endothelial cells and protect them against oxidative injury. In support of this hypothesis, Sakharov et al (50) documented that indirect targeting of catalase mediated by polyclonal anti-endothelial antibody protects cultured endothelial cells against the cytotoxic effects of H2O2. As further progress, several monoclonal and polyclonal antibodies that bind selectively to the vascular endothelium in vitro and in vivo and accumulate in the pulmonary vasculature in various animal species, including humans, after systemic administration have been defined (9, 39, 43). The present study focused on the utilization of two monoclonal antibodies that recognize the constitutive endothelial surface antigens ACE (anti-ACE MAb 9B9) and ICAM-1 (anti-ICAM-1 MAb 1A29). The lungs contain 20-30% of the total number of endothelial cells in the body, and, therefore, pulmonary tissue is enriched in these antigens. Both anti-ACE MAb 9B9 and anti-ICAM-1 MAb 1A29 react with rat antigens (9, 43). Epitopes for both antibodies are abundant in the rat lung vasculature (Fig. 1). Both anti-ACE and anti-ICAM-1 antibodies circulating in the perfused rat lungs accumulate in the lung tissue to a similar extent (Fig. 2A). Catalase conjugated with either antibody also accumulates in the perfused rat lungs: up to 20% of antibody-conjugated catalase accumulated in the lung tissue (Fig. 2B).
Results for the specific pulmonary uptake of MAb 9B9- or MAb 1A29-conjugated 125I-catalase coincide with previous data by Muzykantov et al. (37) obtained in rats in vivo. Although encouraging, these data, however, did not provide the answer to the major question: does immunotargeting of catalase afford significant augmentation of antioxidant defense for the endothelium? To address this question, we utilized a model of isolated rat lung perfused with H2O2. This model has been used by several groups (22, 45) for the investigation of the mechanisms of an oxidative injury to the pulmonary vasculature. In accordance with data published by these groups, the addition of H2O2 into the lung perfusate led to findings compatible with the elevation in pulmonary vascular permeability as indicated by an elevation in the wet-to-dry lung weight ratio and the pulmonary arterial and tracheal pressures as well as an elevation in ACE activity in the perfusate of isolated lungs (22, 45).
Elevation in ACE activity in the perfusate could reflect either stimulation of ACE shedding from the endothelium and/or desquamation of endothelial cells or membrane vesicles. ACE-expressing cells physiologically shed ACE from the plasma membrane via proteolytic cleavage by a specific membrane-associated protease (secretase) (48). Proteolytic shedding should lead to an elevation in the hydrophilic form of ACE in the perfusate because cleaved ACE lacks a hydrophobic transmembrane domain. In contrast, cellular or particulate desquamation should lead to the appearance of the hydrophobic (membrane-anchored) form of ACE in the perfusate. Based on these considerations, we discriminated between ACE shedding and cellular and/or particulate desquamation by determining the distribution of the perfusate ACE activity between the aqueous and detergent phases. Our results document that perfusion with H2O2 leads to the appearance in the perfusate of cellular particles containing hydrophobic ACE (Fig. 4). This result corroborates a recent observation (44a) that oxidants induce release of plasma membrane vesicles from endothelial cells in culture. Therefore, our results show that oxidative insult in the whole organ leads to both stimulation of ACE shedding and desquamation of membrane particles.
On the basis of the determination of ACE activity in the perfusate, catalase conjugated with either ACE or ICAM-1 MAb provides ~60% protection against endothelial injury induced by 2 or 5 mM H2O2. The similar effectiveness of catalase conjugated with anti-ACE and anti-ICAM-1 MAbs indicates that the protective effect of catalase conjugated with anti-ACE MAb 9B9 is not antigen specific. In other words, protective suppression of H2O2-induced ACE release by 1A29-Cat implies that, in case of 9B9-Cat, this effect is not mediated by interaction of anti-ACE MAb 9B9 with endothelial ACE. This consideration is important in the light of previous observations made by Caldwell et al. (8) that antibodies against ACE may alter its membrane metabolism and rate of its shedding from the plasma membrane. Noteworthy, antibody-conjugated catalase not only suppressed the H2O2-induced elevation in total ACE activity in the perfusate but also provided marked normalization of the phase-separation profile of ACE activity (Fig. 9). At a physiological level, targeting of catalase affords normalization of the H2O2-induced elevation in tracheal pressure and lung wet-to-dry weight ratio as well as normalization of the delayed elevation in pulmonary arterial pressure. Conjugates failed to block a transient elevation in the pulmonary arterial pressure that occurred immediately after H2O2 infusion (Fig. 6). It is tempting to speculate that H2O2 escaping degradation in the first minutes may act as a secondary messenger in the pulmonary vasculature.
Therefore, our results demonstrate that catalase conjugated with an MAb recognizing surface endothelial antigens not only accumulates in the rat lung but also affords significant augmentation of antioxidative defense to the pulmonary endothelium, detectable by the attenuation of ACE release and prevention of an increase in pulmonary pressure and edema in response to H2O2. The protective effect of the antibody-conjugated catalase is specific because nonconjugated components of the conjugates, as well as IgG-Cat, did not provide any protection. Indeed, IgG-Cat rather aggravated H2O2-induced injury, although nonconjugated IgG, as well as nonconjugated antibodies or catalase, had no detectable effect. Speculatively, this effect may be explained by the interaction of IgG-Cat with residual pulmonary leukocytes. Although we could not detect those in the lung tissue sections, others (53) have reported that preparations of isolated rat lungs may contain residual leukocytes. Because conjugates represent large multimolecular complexes possessing multiple Fc fragments, they may interact multivalently with Fc receptors of residual leukocytes. This notion is indirectly supported by the fact that pulmonary uptake of IgG-Cat is several times higher than that of IgG or catalase alone. IgG-Cat interacting with leukocytes via an immune complex-like pathway may aggravate H2O2-induced injury by release of other oxidants, proteases, and other cytotoxic agents. Presumably, Fc-mediated reactions may play an even more important role in vivo. This is of importance in the context of the safety of conjugate administration. Utilization of the conjugates based on Fab fragments of the carrier antibodies may help solve the potential problems.
Importantly, both carrier antibodies used in the present study, anti-ACE MAb 9B9 and anti-ICAM-1 MAb 1A29, demonstrated their effectiveness for targeting catalase and antioxidative protection by the conjugates. ICAM-1 antibody seems to be an excellent candidate for targeting antioxidants (and, potentially, other drugs) to the pulmonary endothelium challenged with proinflammatory stimuli. Results from our laboratories, as well as from other groups, have documented that endotoxin challenge or systemic complement activation upregulates surface expression of ICAM-1 in the pulmonary endothelium and leads to an increase in the pulmonary uptake of radiolabeled anti-ICAM-1 antibody (34, 35, 42). Therefore, we may expect preferential targeting of antioxidants to the endothelium altered by inflammation, i.e., cells that suffer oxidative injury. Second, anti-ICAM-1 antibodies attenuate tissue injury induced by ischemia-reperfusion in the lung (10) and heart (26), rheumatoid arthritis (23), and pneumonia (11, 25). Thus anti-ICAM-1-catalase might provide a dual therapeutic effect via downregulation of leukocyte adhesion and augmentation of an antioxidative defense. Third, endothelial cells internalize ICAM-1 antibodies at a very slow rate, if they internalize them at all (24, 47). Thus anti-ICAM-1-catalase may be especially effective for protection against extracellular oxidants. For all these reasons, this conjugate is an excellent candidate for protection of the pulmonary endothelium against leukocyte-mediated oxidative injury.
On the other hand, anti-ACE-catalase provides very effective targeting to the normal pulmonary vascular endothelial cells (37). Importantly, in previous studies, Muzykantov and colleagues documented that endothelial cells internalize anti-ACE MAb 9B9 (38) and that radiolabeled catalase conjugated with this MAb undergoes intracellular uptake without marked degradation (37). Thus 9B9-Cat may provide a mechanism for the augmentation of intracellular antioxidative defense in the endothelial cells. This issue may be important in cases of oxidative insults induced by membrane-permeable oxidants (e.g., H2O2) or insults associated with overproduction of oxidants by the endothelium itself. For example, lung ischemia-reperfusion induces oxidative injury to the pulmonary endothelium by a mechanism associated with enhanced generation of oxidants by endothelium (2, 15). Therefore, 9B9-Cat may be appropriate for treatment of ischemia-reperfusion-related oxidative insults (lung transplantation-associated injury and heart-lung bypass-associated injury). Importantly, these cases would provide an opportunity for pretreatment of the pulmonary vasculature with the conjugate.
In conclusion, the present study confirms the validity of a novel strategy for antioxidant protection of the pulmonary endothelium and provides a background for its further exploration in models utilizing intact animals. Immunotargeting of catalase (or a combination of catalase and SOD) seems to be especially appropriate for application in an acute situation [e.g., adult respiratory distress syndrome (29)] and could accompany other approaches in the molecular therapy of vascular diseases (20). Gene therapy could offer augmentation of endothelial antioxidative defense via transfection of the endothelium with genes encoding antioxidant protein catalase (14), metallothionein (52), or endothelial nitric oxide synthase (54). Gene therapy, however, may not be effective during the first hours after intervention because protein synthesis requires time. In contrast, enzymes targeted to the endothelial cells provide immediate protection. Therefore, a combination of enzyme immunotargeting and gene therapy may offer a strategy possessing a broad therapeutic window and high potential effectiveness.
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ACKNOWLEDGEMENTS |
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We thank Dr. S. Albelda for reading the manuscript and helpful discussions, Dr. Donald Buerk for the determination of H2O2 in the perfusate with an H2O2 electrode, and D. Win Harshaw for valuable help with the experiments.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant RO1-HL-41939 (to A. B. Fisher) and American Heart Association Grant-in-Aid 95012700 and Established Investigator Grant 9640204N (to V. R. Muzykantov).
E. Atochina was supported by a Fellowship in Pulmonary Research from the Will Rogers Foundation (White Plains, NY).
Address for reprint requests: V. R. Muzykantov, Institute for Environmental Medicine, Univ. of Pennsylvania School of Medicine, One John Morgan Bldg., 36th St. and Hamilton Walk, Philadelphia, PA 19104-6068.
Received 29 September 1997; accepted in final form 21 May 1998.
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