Vascular immunotargeting of glucose oxidase to the endothelial antigens induces distinct forms of oxidant acute lung injury: targeting to thrombomodulin, but not to PECAM-1, causes pulmonary thrombosis and neutrophil transmigration

Abstract

Oxidative endothelial stress, leukocyte transmigration, and pulmonary thrombosis are important pathological factors in acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Vascular immunotargeting of the H(2)O(2)-generating enzyme glucose oxidase (GOX) to the pulmonary endothelium causes an acute oxidative lung injury in mice.(1) In the present study we compared the pulmonary thrombosis and leukocyte transmigration caused by GOX targeting to the endothelial antigens platelet-endothelial cell adhesion molecule (PECAM) and thrombomodulin (TM). Both anti-PECAM and anti-TM delivered similar amounts of (125)I-GOX to the lungs and caused a dose-dependent, tissue-selective lung injury manifested within 2 to 4 hours by high lethality, vascular congestion, polymorphonuclear neutrophil (PMN) sequestration in the pulmonary vasculature, severe pulmonary edema, and tissue oxidation, yet at an equal dose, anti-TM/GOX inflicted more severe lung injury than anti-PECAM/GOX. Moreover, anti-TM/GOX-induced injury was accompanied by PMN transmigration in the alveolar space, whereas anti-PECAM/GOX-induced injury was accompanied by PMN degranulation within vascular lumen without PMN transmigration, likely because of PECAM blockage. Anti-TM/GOX caused markedly more severe pulmonary thrombosis than anti-PECAM/GOX, likely because of TM inhibition. These results indicate that blocking of specific endothelial antigens by GOX immunotargeting modulates important pathological features of the lung injury initiated by local generation of H(2)O(2) and that this approach provides specific and robust models of diverse variants of human ALI/ARDS in mice. In particular, anti-TM/GOX causes lung injury combining oxidative, prothrombotic, and inflammatory components characteristic of the complex pathological picture seen in human ALI/ARDS.

Figure 1.

Binding of TM and PECAM antibodies in the murine lungs. Top: In situ immunostaining of lung frozen sections (A–C). Bottom: Detection of intravenously injected anti-TM and anti-PECAM mAb (D and E). Binding of anti-TM and anti-PECAM was revealed by secondary peroxidase-conjugated antibodies. Note that anti-PECAM and anti-TM binds to antigen both in situ and in vivo. Antigens are detected on pulmonary capillaries (see insets in bottom panels), and larger pulmonary vessels (V). Original magnifications: ×200 (A–F); ×500 (d, e).

Figure 2.

Pulmonary targeting of GOX to TM and PECAM-1. The level of ¹²⁵I in blood and lung 1 hour after intravenous injection of IgG/¹²⁵I-GOX (filled bars), anti-TM/¹²⁵I-GOX (open bars), and anti-PECAM/¹²⁵I-GOX (hatched bars) in intact mice. The data are shown as mean ± SEM (n = 4). The pulmonary uptake of anti-TM/¹²⁵I-GOX and anti-PECAM/¹²⁵I-GOX was significantly higher than that of IgG/¹²⁵I-GOX (P < 0.01).

Figure 3.

Survival of mice injected with anti-TM/GOX (A) or anti-PECAM/GOX (B). Mice were injected with indicated doses of conjugate and the survival was determined during the 24 hours after injection. The figure presents cumulative results of several series of experiments. Number of animals for each conjugate dose is indicated.

Figure 4.

Dose-dependent lung injury after injection of anti-TM/GOX. Left column: The dose of anti-TM/GOX injected (0, 25, 50, and 75 μg) and a typical ALIS observed at this dose. Middle: The panels show typical gross morphological view of the lungs at postmortem. Right: The panels represent representative histopathological sections obtained from animals injected with the corresponding dose of anti-TM/GOX, stained with H&E (original magnification, ×100). Lungs from saline-injected control mice appeared evenly pink and without cellular infiltrates or vascular congestion on microscopic examination (ALIS 1, no detectable injury). Injection of 25 μg of anti-TM/GOX produced faintly red lungs (ALIS 4) with microscopic vascular congestion and mild edema. Injection of 50 μg of anti-TM/GOX resulted in frankly red and boggy lungs (ALIS 7) and microscopic signs of severe vascular congestion, proteinaceous fluid in the alveoli, moderate to severe alveolar hemorrhage, and inflammatory infiltration. Injection of 75 μg of anti-TM/GOX induced severe injury manifested by almost black hemorrhaged lung surface, tensely swollen lungs (ALIS 10), characterized microscopically by florid pulmonary edema, diffuse alveolar hemorrhage, and an intense acute inflammation.

Figure 5.

Comparison of the injuring potency of anti-TM/GOX and anti-PECAM/GOX. The lethality (A) and severity of lung injury (ALIS, B) were determined after intravenous injection of indicated dose of anti-TM/GOX (circles) or anti-PECAM/GOX (squares) in intact mice. The ALIS data are shown as mean ± SEM for each group, with number of animals per group varying from 6 to 58. Note that in the intermediate doses range (20 to 70 μg of GOX/mouse), anti-TM/GOX causes higher lethality and inflicts more severe lung injury than anti-PECAM/GOX.

Figure 6.

Anti-TM/GOX and anti-PECAM/GOX induce pulmonary edema in mice. The lung wet-to-dry ratio (A) and protein level in the BAL fluid (B) have been determined 4 hours after intravenous injection of saline (control) or equally potent doses of the conjugates [35 μg of anti-TM/GOX (n = 24) or 70 μg of anti-PECAM/GOX (n = 9)] in intact mice. The data are shown as mean ± SEM, P < 0.001 versus control (asterisk).

Figure 7.

Detection of products of tissue oxidation in the lungs. Mice were injected with saline (control, top) or injuring doses of anti-PECAM/GOX (αPECAM, middle) or anti-TM/GOX (αTM, bottom). The lung tissue sections were stained with antibody directed against 8-epi iPF_2a-III F₂ isoprostane, a marker of lipid peroxidation (left column, 8-epi), antibody to nitrotyrosine, a marker of protein oxidative nitration (middle column, Nitro-Y), or control rabbit IgG (control, right column). The positive immunostaining was revealed by secondary antibody conjugated with alkaline phosphatase (blue color), Neutral Red counterstaining was used to mark individual cells. Original magnification, ×200.

Figure 8.

Anti-TM/GOX and anti-PECAM/GOX cause pulmonary leukocyte sequestration. A: H&E sections from paraffin-embedded lung tissues harvested 4 hours after injection of saline control (a), or equally injuring doses of anti-TM/GOX (b) or anti-PECAM/GOX (c). B: Analysis of MPO in the lung tissue homogenates after injection of a marginally effective dose of either conjugate (left pair, low dose: 15 mg of anti-TM/GOX and 30 mg of anti-PECAM/GOX) or highly injuring dose of either conjugate (right pair, high dose: 30 mg of anti-TM/GOX and 75 mg of anti-PECAM/GOX). The data in B are shown as mean ± SEM, dotted line represents baseline value.

Figure 9.

Leukocyte dynamics in the lung differ after injection of anti-TM/GOX and anti-PECAM/GOX. Total WBC counts in the BAL fluid (A) reveals that only anti-TM/GOX (squares), but not anti-PECAM/GOX (circles) cause WBC influx into the alveoli. B: Typical BAL cytospin preparations at an original magnification of ×500 after injection of saline control (a), anti-PECAM/GOX (b), or anti-TM/GOX (c). C shows the number of PMN neutrophils in BAL obtained 4 hours after injection of the indicated conjugates. The data are shown as means ± SEM.

Figure 10.

Quantitative evaluation of neutrophil compartmental localization in control and GOX/Ab-treated lungs. PMN were evaluated in 10 high-power light-microscopic fields (×100 oil objective) from H&E lung sections. A: Total number of neutrophils in whole-lung sections from untreated baseline (control) animals and maximally injured, GOX-immunoconjugate treated (ALIS = 10) mice. B: PMN in capillaries (black) or alveoli (checkered) in control or GOX-treated mice. C: Percent increase of neutrophil numbers in the two compartments after targeted GOX treatment over baseline, untreated mice (means ± SEM).

Figure 11.

Different ultrastructural features of lung injury induced by anti-TM/GOX and anti-PECAM/GOX in mice. Transmission electron micrographs show typical morphological patterns of the lungs after injection of anti-TM/GOX (A) or anti-PECAM/GOX (B). A: Lower power (original magnification, ×4,400): visible blebbing of epithelial cells, swelling of endothelium and proteinaceous material (asterisk) with fibrin strands (arrowheads) in the alveoli. Capillaries are packed with PMN neutrophils and platelets. Inset (a, high power ×15,000) shows leukocytes retaining granules (g) in extravascular space and focal damage of endothelial cell (double arrows). B: Low power (original magnification, ×4,400): severe interstitial edema (Ed), proteinaceous alveolar material, numerous PMNs trapped in vessels contain no visible granules and condensed, pycnotic nuclei. Inset (b, high power ×15,000) shows neutrophil in capillary, with condensed nuclear chromatin, dense cytoplasm, and loss of granules.

Figure 12.

Anti-TM/GOX causes profound pulmonary thrombosis. Transmission electron micrographs and H&E staining (insets; original magnifications, ×500) of lung tissue after anti-TM/GOX (A, a) and anti-PECAM/GOX (B, b) injection in mice. A, a: A venule occluded by platelet (PL) thrombus with entrapped PMNs and red blood cells (RBCs). B, b: Absence of thrombi in either arteriole or capillaries. Multiple degranulated PMNs are visible in capillaries. Original magnifications: ×4000 (A); ×7700 (B); ×250 (a and b). Alv, alveolus; V, venule; PL, platelet.

Figure 13.

Detection of platelet thrombi in GOX-treated lungs. Mice were given injuring doses of anti-TM/GOX or anti-PECAM/GOX and lungs were harvested after 4 hours. Lung sections were processed for immunohistochemical platelet detection using an anti-CD 41 (anti IIb Ab). Platelets and platelet thrombi are shown as red-brown staining. A: Control, untreated tissues, given no primary Ab. B: Control untreated lung stained with anti-CD41. C and D: Lung sections treated with either anti-PECAM/GOX (C) or anti-TM/GOX (D), stained with anti-CD41. Original magnifications, ×250X.