The lung is a common target in systemic vasculitides associated with antineutrophil cytoplasmic antibodies (ANCA). In the present study, we tested the hypothesis that the presence of antibodies directed against myeloperoxidase (MPO) induces pulmonary (vasculitic) lesions when neutrophils release lysosomal enzymes. Brown Norway (BN) rats were immunized with human MPO in complete Freund's adjuvant (CFA) or with CFA alone. Two weeks after immunization, rats had developed antibodies to human and rat MPO. Next, isolated single left lung perfusion was performed with human neutrophil lysosomal extract containing MPO and proteolytic enzymes. Rats were killed at 15 min, 4 h, and 10 d after perfusion. Tissue samples from the left and right lung were examined for vasculitic lesions and inflammatory cell infiltrates. At 15 min and 4 h, left lungs from control and MPO-immunized rats showed a mild influx of polymorphonuclear cells. At 10 d, patchy inflammatory cell infiltrates, consisting predominantly of polymorphonuclear leukocytes (PMNs) and monocytes, were observed throughout the parenchyma of the left lung in MPO-immunized rats. Occasionally, granuloma-like lesions, giant cells, and foci of alveolar hemorrhage were observed as well. Far less severe lesions were seen in control immunized rats. Strikingly, at 10 d after perfusion, severe pulmonary tissue injury was observed also in right lungs from MPO-immunized rats whereas right lungs from control immunized rats appeared normal. The lesions were characterized by influx of PMNs and monocytes and, in some rats, foci of alveolar hemorrhage. These studies suggest that the presence of an anti-MPO directed autoimmune response contributes to generalized pulmonary tissue injury after local release of products of activated neutrophils, which supports a pathogenic role of MPO-ANCA.
Antineutrophil cytoplasmic antibodies (ANCA) are autoantibodies observed in patient serum in a wide range of vasculitic diseases including Wegener's granulomatosis, Churg-Strauss syndrome, idiopathic crescentic glomerulonephritis, and microscopic polyangiitis where a renal–pulmonary syndrome including pauci-immune necrotizing crescentic glomerulonephritis (NCGN) and pulmonary capillaritis can be observed (1).
Proteinase 3 and myeloperoxidase (MPO), both myeloid lysosomal enzymes, have been identified as the primary target antigens for ANCA in those disorders (2-5). It has been well established that detection of ANCA is useful in the diagnosis and monitoring of systemic vasculitic disorders (1). However, the pathophysiological role of ANCA in their associated diseases has not been fully elucidated. In vitro experiments have shown that ANCA can activate neutrophils leading to oxygen radical production and degranulation of proteolytic lysosomal enzymes (6-8). In addition, ANCA promote the adhesion of neutrophils to monolayers of endothelial cells and may cause vascular injury by activating primed neutrophils to induce endothelial cell lysis (9-11). Although these in vitro observations suggest a pathogenic role for ANCA, their in vivo relevance is not clear. Studying the pathophysiological role of ANCA in vivo we previously reported on an animal model for anti-MPO-associated pauci-immune NCGN. In this model, pauci-immune NCGN was induced in MPO-immunized rats by renal perfusion of MPO, proteolytic enzymes, and H2O2 (12).
In most cases of ANCA-associated vasculitides, disease manifestations are not confined to the kidneys (13). Especially pulmonary involvement has frequently been reported (14). In the present study, we tested the hypothesis that in ANCA-associated vasculitis a similar pathogenesis may underlie the disease manifestations in both lungs and kidneys. To this end, an animal model for anti-MPO-associated pulmonary vasculitis was developed in analogy to the kidney model. In this model Brown Norway (BN) rats were immunized with human MPO and a single left lung perfusion with a neutrophil lysosomal extract was performed.
All experiments were performed in specific-pathogen-free (SPF), 3-mo-old BN rats, fed ad libitum with standard chow (Hope Farms, Woerden, the Netherlands).
Human polymorphonuclear leukocytes (PMN) were isolated from buffy coats on a ficoll density gradient followed by dextran sedimentation. After sedimentation, the remaining erythrocytes were removed by hypotonic lysis. MPO was extracted from PMN by dissolution of the cells in cetyltrimethyl ammonium bromide (CETAB; Sigma Chemical Co., St. Louis, MO) and sonification. Nuclei and membrane fragments were discarded by ultracentrifugation and the extract was absorbed to a concanavalin A Sepharose gel (Pharmacia, Uppsala, Sweden) and eluted with α-methyl-d-mannoside (Sigma). The ratio between the optical density (OD) of this eluate obtained at 428 nm (showing a specific spectral band for MPO) and the OD obtained at 280 nm was measured. Eluted fractions with a ratio (OD 428/280) greater than 0.5 were pooled and extensively dialyzed against sodium acetate buffer pH 6.0. The resulting extract had an OD 428/280 ratio greater than 0.5 and contained mainly MPO, proteinase 3, and trace amounts of elastase, but no lactoferrin as determined by sandwich ELISA. Specific bands for MPO (15, 39, and 58 kD) and proteinase 3 and elastase (± 30 kD) were found by gel electrophoresis (data not shown).
For immunization and ELISA, MPO was further purified on a Sephadex G100 gel (Pharmacia). The final MPO preparation had an OD 428/280 ratio of 0.85. The preparation was not contaminated with proteinase 3, elastase, or lactoferrin as estimated by antigen-specific ELISA. Analysis by gel electrophoresis showed specific bands for human MPO (at 15, 39, and 58 kD). Rat MPO was purified from rat peritoneal exudate cells as described (15).
BN rats were immunized with human MPO or control solution (sodium acetate buffer), both in complete Freund's adjuvant (CFA) supplemented with 5 mg/ml H37Ra (Difco, Detroit, MI). Rats received 10 μg MPO or control solution (sodium acetate buffer) without MPO subcutaneously in 0.2 ml at two sites near the base of the tail.
Cytospin slides were prepared form rat peritoneal exudate cells as described previously (15). Slides were fixed in 100% ethanol for 10 min. After incubation for 20 min with normal goat serum (10%), the slides were incubated with rat sera (1:10) for 1 h. After extensive washing in phosphate-buffered saline (PBS), slides were incubated with fluorescein isothiocyanate (FITC)-labeled goat anti-rat Ig (Dakopatts, Glostrup, Denmark) for 1 h. Slides were mounted in glycerol/PBS solution (Citifluor; Citifluor Ltd, London, UK).
Labstar (Greiner, Kresmünster, Austria) 96-well microtiter plates were coated 2 h at 37° C with purified human or rat MPO at protein concentration of 2 μg/ml in 0.1 M carbonate buffer, pH 9.6. The plates were incubated for 1 h at 37° C with rat sera, diluted in 0.01 M Tris, 0.05% Tween, 1% bovine serum albumin (BSA), 0.3 M NaCl, pH 8.0, and 1% normal goat serum, starting at a dilution of 1:100 for human MPO and 1:25 for rat MPO. Antibody binding was detected with alkaline phosphatase–labeled goat affinity purified antibody to rat IgG (Cappel Laboratories, West Chester, PA) followed by p-nitrophenyl phosphate disodium as a substrate (Sigma). The reaction was stopped with sodium hydroxide. The OD was read at 405 nm, and a standard curve was prepared from a reference serum. Antibody concentrations were computed from the linearized titration curve obtained after log-logit transformation of the absorbances of the respective dilutions of the reference serum. The concentration of the reference serum was arbitrarily set at 100 U.
Two weeks after immunization unilateral perfusion of the left lung was performed according to a modification of the method described by Weksler and coworkers (16). Rats were anaesthetized with halothane, O2, and N2O, and ventilated (Harvard Rodent ventilator; Harvard Apparatus Ltd, Edenbridge, UK) via an endotracheal tube (17). A left thoracotomy was performed in the fifth intercostal space and the pulmonary artery and vein were exposed and carefully dissected as described previously for lung transplantation (17). Clamps were temporarily put on the pulmonary artery and on the pulmonary vein, close to the mediastinum, isolating the left lung from the blood circulation. A cannula, first flushed with heparin, was inserted into the pulmonary artery through a small incision and secured with sutures for the inflow of the perfusate. The two clamps were then removed and the lung was allowed to reperfuse in order to reduce ischemia time. After 10 min, the clamps were replaced identically and a small incision was made in the pulmonary vein to allow blood and perfusion fluids to escape. To estimate the volume of the perfusion fluids needed to perfuse the whole lung, left lungs of two rats were perfused as previously described with a solution of Evan's Blue. These pilot experiments showed that the entire lung stained blue after perfusion of 4 to 5 ml of dye solution and indicated a uniform distribution of the perfusate. Based on these results, experiments were set up in which the lung was flushed with PBS/heparin and perfused using infusion pumps (2.5 ml/min, room temperature) according to the following sequence: (1) PBS 10 ml, (2) neutrophil lysosomal extract in 4 ml PBS (400 μg of protein containing approximately 200 μg of MPO) which was allowed to react for two min, (3) PBS 10 ml. During the procedure, the thoracic cavity was extensively washed with PBS to avoid a toxic effect of the PMN lysosomal extract on the pleura. After perfusion, the artery cannula was removed and the incisions closed with one or two stitches. The two clamps were removed and the lung was allowed to reperfuse. Total procedure time was about 2 h. During the procedure, the left lung was continuously ventilated.
At 15 min, 4 h, or 10 d rats were anesthetized with pentobarbital intraperitoneally and underwent a tracheotomy. A median thoracotomy was performed, the left atrium opened, the right ventricle cannulated and perfused with PBS under low pressure (15 cm water pressure) until the lungs became completely pale. The lungs and heart were then carefully removed from the thorax. After removal of heart, vessels, and esophagus, the lungs were instilled with 5 ml of embedding compound (Tissue Tek; Sakura Finelek, Europe, Zoeler-Waude, The Netherlands) to preserve optimal morphology.
Cross-sections of whole right and left lungs were performed with razor blades. One half of each lung was fixed in 2% paraformaldehyde/PBS for light microscopy and the remaining tissue immediately frozen in liquid nitrogen for immunohistological staining.
Paraformaldehyde-fixed tissues were embedded in paraffin and 3-μm sections were stained with hematoxylin–eosin. For frozen tissues, 7-μm-thick sections were cut on 3-AminoPropyl-3-ethoxy-Silane (2% in acetone; Sigma) coated slides. Sections were fixed with acetone (100%). After fixation, sections were air dried and then incubated with 10% normal rabbit or goat serum in PBS, depending on the conjugate to be used. PMNs, monocytes, and T cells were detected by immunoperoxidase staining using monoclonal antibodies HIS48 (18), R73 (19), ED1 (Serotec), respectively, followed by peroxidase-labeled rabbit anti-mouse antibodies (Dakopatts). We used aminoethylcarbimazole and H2O2 as the substrate. The endogenous peroxidase activity was blocked with 0.05% H2O2. All dilutions were made in PBS. Sections were counterstained with Mayer's hematoxylin. A semiquantitative scoring system was used to evaluate lung injury on paraffin sections (20):
1. Number of macroscopically discernible spots of inflammation: absent = 0; < 5 = 2; 5 to 10 = 3; > 10 = 4.
2. Thickened alveolar walls: absent = 0; present = 1.
3. Granulomas: < 25% = 1; 25 to 50% = 2; 50 to 75% = 3; > 75% = 4 of total lung section.
4. Leukocyte infiltrates: few = 1; moderate = 2; many = 3.
5. Hemorrhage: absent = 0; present = 2.
All sections were scored blindly. For each animal, an overall injury score was calculated by adding the individual scores.
All results are expressed as the mean ± SD and were analyzed using the two-tailed Mann-Whitney U test.
Two groups of animals were studied. Group 1: BN rats immunized with human MPO and perfused with the neutrophil lysosomal extract. Rats were killed at 15 min (n = 2), 4 h (n = 1), and 10 d (n = 7) after perfusion. Group 2: Control immunized BN rats perfused with the neutrophil lysosomal extract. Rats were killed at 15 min (n = 2), 4 h (n = 1), and 10 d (n = 7) after perfusion.
By indirect immunofluorescence staining, sera from MPO- immunized rats showed a positive staining on cytospin preparations of rat neutrophils (Figure 1A). No staining was observed with sera from control immunized animals. As determined by ELISA, all rats immunized with human MPO developed antibodies against human and rat MPO after 2 wk (Figures 1B and 1C). Control immunized rats were consistently negative.
No mortality occurred in the 20 animals studied. The mean ischemia time of the perfused left lung was 39.0 min ± 5.0 for MPO-immunized animals and 35.0 min ± 4.0 for control immunized animals (not significantly different).
Initial experiments were performed to test the feasibility of the perfusion procedure. Therefore, left lungs of control and MPO-immunized rats were perfused with PBS. At 10 d after perfusion no significant pulmonary injury was observed upon histologic examination in either the left or right lung in these rats (Figure 3A). At 15 min or 4 h after perfusion of the PMN lysosomal extract, macroscopic examination of the left lungs obtained from MPO or control immunized rats revealed large areas of atelectasis. Right lungs appeared normal macroscopically.
By light microscopy, no significant lesions were found in the left lungs of control immunized rats killed at 15 min after perfusion. At 4 h, a mild influx of HIS48+ PMNs was observed (Figures 2A and 5A). Left lungs of MPO-immunized rats killed at 15 min also appeared normal although in one rat foci of alveolar hemorrhage were observed. At 4 h, increased cellularity of the alveolar capillaries was found owing to influx of PMNs, at some places, associated with alveolar hemorrhage (Figures 2B and 5B). Right lungs of control and MPO-immunized rats showed a normal morphology at these time points although a slight increase in HIS48+ PMNs was observed at 4 h.
At 10 d after perfusion, patchy granulomatous-like inflammatory lesions were observed throughout the lung parenchyma in left lungs of all MPO-immunized rats (Figure 3C). The lesions consisted of inflammatory cell infiltrates, predominated by HIS48+ PMNs and ED1+ monocytes, within the pulmonary interstitium associated with small arteries and alveolar capillaries (Figures 5E and 5F). Low numbers of R73+ T cells were also observed. In all MPO-immunized rats, the inflammatory lesions occupied more than 50% of the lung tissue whereas in five out of seven rats more than 75% of the lung tissue was involved. Granuloma-like lesions and giant cell formation were occasionally observed, and, at some places, tissue necrosis and abscess formation was found (Figures 3D and 3E).
At 10 d after perfusion of the neutrophil lysosomal extract, left lungs of control immunized rats also showed inflammatory lesions (Figure 3B). The histopathological characteristics of these lesions were comparable to those from MPO-immunized rats including patchy inflammatory lesions and interstitial expansion and edema (Figures 3B, 5C, and 5D). The overall tissue injury score tended to be different between MPO− and control immunized rats (9.4 ± 1.7 versus 6.9 ± 2.7, respectively, p < 0.07, Figure 6).
At 10 d after perfusion of the left lung with the neutrophil lysosomal extract, right lungs from control immunized rats showed normal morphology and no extensive inflammatory lesions were observed (Figure 4A). In contrast, patchy inflammatory lesions were found in right lungs from MPO-immunized rats consisting of infiltrates of PMNs and monocytes (Figure 4B). The area of lung tissue involved ranged from < 25% in four rats to > 75% in three rats. In two of the seven rats, foci of hemorrhage were present (Figure 4C). The mean tissue injury score was 5.6 ± 2.4 which was significantly higher than that of control immunized rats (0.9 ± 0.7, p < 0.02).
In systemic vasculitides associated with ANCA, pulmonary involvement is frequently observed. Disease manifestations in the lungs are found in patients positive for either antiproteinase 3 antibodies or anti-MPO antibodies although some studies have suggested that microvascular lung injury is more often associated with anti-MPO antibodies (14, 21, 22). In lung biopsies, a variety of histologic injury patterns can be observed but intra-alveolar hemorrhage with necrotizing capillaritis is one of the most common histopathological findings (13, 23, 24). The close association of ANCA with systemic vasculitis suggests that these autoantibodies may play a role in the pathophysiology of their associated diseases. Evidence supporting this hypothesis has been provided by several in vitro studies. These studies have shown that ANCA are capable of activating cytokine-primed PMNs leading to the production of reactive oxygen species and the release of lysosomal enzymes including the ANCA antigens themselves (6-8). The primary ANCA antigens, proteinase 3 and MPO, are cationic proteins which upon release from PMNs may bind to negatively charged structures such as basement membranes and the luminal side of endothelial cells. Subsequently, ANCA may bind leading to the formation of local immune complexes. These local immune complexes may aggravate the inflammatory response by activation of the complement system and enhancement of inflammatory cell influx. Based on this hypothesis, our group previously developed an animal model for anti-MPO-associated NCGN (12). In this model, anti-MPO-associated NCGN was induced in BN rats, previously immunized with human MPO, by renal perfusion of PMN lysosomal enzymes including MPO and its substrate H2O2 (12).
In the present study, we hypothesized that, in the setting of ANCA-associated vasculitis, a similar pathogenesis may underlie disease manifestations in kidneys and lungs. Therefore, experiments were designed in which, in analogy to the kidney model, PMN lysosomal enzymes were perfused into the left lung of BN rats previously immunized with human MPO or control solution. At 10 d after perfusion, extensive lung injury was observed in left lungs of MPO-immunized rats consisting of patchy inflammatory cell infiltrates, interstitial expansion, and, occasionally, granuloma-like lesions and giant cell formation. At this time point, left lungs of control immunized rats also showed pulmonary injury with similar histopathological characteristics as observed in left lungs of MPO-immunized rats. As determined on a semiquantitative scoring system for tissue injury, the pulmonary lesions in control rats tended to be less severe compared with MPO-immunized rats. Histopathological examination of the right lungs at 10 d after perfusion revealed the presence of patchy inflammatory lesions in MPO-immunized rats. In contrast, no inflammatory lesions were observed in right lungs of control immunized rats at this time point.
The observation of pulmonary injury in left lungs of control immunized rats after perfusion of a PMN lysosomal extract via the pulmonary vasculature indicates a direct toxic effect of the PMN extract on lung tissue. The PMN extract consists of MPO and active proteolytic lysosomal enzymes such as elastase and proteinase 3 all of which may cause tissue injury (25). In vitro studies have shown that proteinase 3 and elastase are capable of degrading several constituents of basement membranes including heparan sulfate proteoglycan, laminin, and collagen type IV (25). In the lungs, both enzymes have been shown to cause emphysema upon intratracheal insufflation (26). Besides proteolytic enzymes, MPO-induced formation of reactive oxygen radicals may also have contributed to tissue injury. Reaction of MPO with its substrate H2O2 leads to the formation of the highly reactive hydroxyl radical and, in the presence of a halide (chloride, bromide, iodide) produces highly toxic species such as hypochlorous acid (27, 28). Furthermore, it has been demonstrated that intratracheal instillation of MPO and H2O2 causes PMN-independent acute pulmonary injury progressing to interstitial fibrosis within 2 wk (29). As described previously, the experimental model of anti-MPO-associated NCGN included perfusion of a PMN lysosomal extract and H2O2 (12). However, in a subsequent study it was shown that perfusion with H2O2 could be replaced by introducing renal ischemia/reperfusion injury through clamping of the renal artery (30). From these studies it was suggested that ischemia induces endothelial cells to produce H2O2 and, as such, provide the substrate for MPO. In the present study, the ischemia time for the left lung was approximately three times as long compared with the kidney perfusion model. Therefore, perfusion with H2O2 was omitted.
Pulmonary tissue injury in left lungs of MPO-immunized rats at 10 d after perfusion of the PMN lysosomal extract tended to be more severe as compared with left lungs from control immunized rats. This suggests that pulmonary injury after perfusion of a PMN lysosomal extract is aggravated in the presence of anti-MPO antibodies, possibly by their capacity to activate primed leukocytes comparable to their potential to aggravate experimental anti-glomerular basement membrane disease in the kidney (15).
A striking observation in the present study is the occurrence of severe inflammatory lesions in right lungs of MPO-immunized rats at 10 d after isolated left lung perfusion of the PMN lysosomal extract. The lesions showed histopathological characteristics similar to those observed in the left lungs but were less extensive. At present, the mechanisms responsible for the development of inflammatory lesions in the right lungs are unknown but several explanations may be considered.
It may be suggested that part of the PMN lysosomal extract has leaked into the right lung at the time of or after perfusion of the left lung. However, the separate vascularization of the left and right lung argues against this explanation. In addition, we recently reported on the development of necrotizing vasculitis in lungs and gut upon systemic administration (via the right jugular vein) of a PMN lysosomal extract and H2O2 in MPO-immunized rats (20). In the present study, however, the left lung was extensively washed before reperfusion to avoid release of the PMN lysosomal extract into the circulation. Moreover, no inflammatory changes were observed in right lungs of control immunized rats. During inflammation large amounts of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), and interferon gamma (IFN-γ) are produced. Release of such inflammatory mediators into the circulation may cause priming of circulating leukocytes, especially PMNs and monocytes, leading to upregulation of adhesion molecules and expression of MPO on the cell surface. Moreover, upregulation of adhesion molecules on the endothelium may occur. In the presence of autoantibodies directed against MPO this may lead to further activation of PMNs and monocytes adherent to the endothelium and may cause tissue injury. As demonstrated here and in previous studies, BN rats develop antibodies to human MPO which cross-react with rat MPO upon immunization with human MPO (15, 20). In addition, studies in dogs have demonstrated that 2 h of warm unilateral lung ischemia followed by reperfusion results in pulmonary tissue injury in the ischemic but also in the nonischemic lung (31). In these studies, tissue injury in the nonischemic lung was characterized by increases in extravascular density and permeability, and neutrophil infiltration (31). Thus, the combination of the systemic effects of inflammatory cytokines released from the inflammatory lesions in the left lung, ischemia-induced influx of PMNs, and the presence of autoantibodies directed against rat MPO may have caused the development of pulmonary injury in the right lungs of MPO-immunized rats. This may be relevant in view of the association between infection and disease activity in ANCA-associated systemic vasculitis (32). Further studies are needed to clarify the exact mechanisms involved.
In summary, our studies suggest that the presence of an anti-MPO directed autoimmune response contributes to generalized pulmonary tissue injury after local release of products of activated neutrophils. As such, the in vivo findings are supportive of a pathogenic role of MPO-ANCA in the pulmonary manifestations of MPO-ANCA-associated diseases.
Supported by a grant from La Société de Pneumologie de Langue Française, Paris, France and by Grant C91.1178 from the Dutch Kidney Foundation.
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