Sialyl Lewisx hybridized complement receptor type 1 moderates acid aspiration injury

Constantinos Kyriakides1, Yong Wang1, William G. Austen Jr.1, Joanne Favuzza1, Lester Kobzik2, Francis D. Moore Jr.1, and Herbert B. Hechtman1

Departments of 1 Surgery and 2 Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The potentially enhanced anti-inflammatory effects of the sialyl Lewisx (sLex)-decorated version of soluble complement receptor type 1 (sCR1) in moderating acid aspiration injury are examined. HCl was instilled in tracheostomy tubes placed in mice, and extravasation of 125I-labeled albumin in bronchoalveolar lavage (BAL) fluid was used to calculate the vascular permeability index (PI). Neutrophil counts in BAL fluid and immunohistochemistry were performed. PI was moderated by 82% after treatment with sCR1sLex compared with 54% in sCR1-untreated mice (P < 0.05). Respective reductions in PI in mice treated 0.5 and 1 h after acid aspiration with sCR1sLex of 70 and 57% were greater than the decreases in PI of 45 and 38% observed in respective sCR1-treated groups (P < 0.05). BAL fluid neutrophil counts in sCR1sLex-treated mice were significantly less than those in sCR1-treated animals, which were similar to those in untreated mice. Immunohistochemistry stained for sCR1 only on the pulmonary vascular endothelium of sCR1sLex- but not sCR1-treated mice. In conclusion, sCR1sLex moderates permeability by antagonizing complement activation and neutrophil adhesion. Delayed complement and neutrophil antagonism significantly reduces injury.

pneumonia; murine; inflammation; complement activation; selectins


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ASPIRATION OF ACIDIC GASTRIC contents leads to acute lung injury and is a potentially serious complication in trauma and surgical patients. Furthermore, it is the leading cause of the adult respiratory distress syndrome after elective surgery (5). The reported mortality after major aspiration ranges between 30 and 94% (1, 5, 20). The pulmonary injury has been described as biphasic (10). There is an early and direct chemical airway injury characterized by copious, protein-rich bronchial secretions, resulting in neutralization of the acid (1). This is followed by a delayed humoral (complement, cytokine, eicosanoid) and cellular (neutrophil, macrophage) response, leading to distal alveolar injury and increased capillary leak (4, 6, 7, 13, 26).

Both the complement system and the polymorphonuclear leukocyte (PMN) have been shown to be important mediators of acid aspiration injury (13, 26). Complement inhibition with the C3b and C4b antagonist soluble complement receptor type 1 (sCR1) has been shown to reduce pulmonary permeability (13, 26). More specifically, it is activation of the alternative complement pathway that leads to increased pulmonary permeability, demonstrated by protection of C3 but not C4 genetic knockout mice against injury (26).

Neutrophil adhesion to the microvascular endothelium is thought to be a prerequisite in the sequence of events leading to PMN sequestration, diapedesis, and release of cytotoxic proteases and oxygen-derived free radicals within injured tissues (14). The adhesion sequence of events starts with leukocyte rolling and tethering on the activated endothelial cell, which is facilitated by the selectin family of adhesion molecules (24). Two adhesion molecules of this family expressed during endothelial cell activation are P- and E-selectin. These can bind to a glycoprotein counterreceptor expressing sialyl Lewisx (sLex)-related oligosaccharides such as P-selectin glycoprotein ligand 1 (PSGL-1), which is present on neutrophils and other leukocytes (9, 15, 22).

In this study of experimental acid aspiration, the protective effects of the sLex-decorated version of sCR1 are examined. It is hypothesized that treatment with sCR1sLex presents a novel means by which complement inhibitory action is combined with antiselectin activity, leading to better protection against injury compared with non-sLex-glycosylated sCR1. Furthermore, the potential of sCR1sLex to localize its action on the activated vascular endothelium where selectin upregulation occurs is investigated. Finally, the likely efficacy of treatment after acid aspiration is studied.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Recombinant complement inhibitory proteins. sCR1 and sCR1sLex were provided by AVANT Immunotherapeutics (Needham, MA). sCR1 was produced in the Chinese hamster ovary cell line DUKX B11 and purified as previously described (27). This molecule possesses no sLex on its N-linked oligosaccharides (21). For sCR1sLex production, the expression plasmid coding for sCR1 was used (27). The plasmid pTCSLDHFR*, coding for a mutant mouse dihydrofolate reductase with an abnormally low affinity for methotrexate, was derived from pSV2-DHFR* and cloned into pTCSLneo by direct substitution of the neomycin resistance gene (23). The Chinese hamster ovary cell line Lec11, which expresses the alpha 1-3-fucosyltransferase activity necessary for synthesis of sLex-related oligosaccharides, was contransfected with pTCSLDHFR* together with the plasmid coding for sCR1 (2, 25). Clones grown in medium containing methotrexate were selected for production of high concentrations of sCR1sLex. Carbohydrate analysis of the purified glycoproteins indicated sLex glycosylation of sCR1sLex (18).

Animals. Male C57BL/6 mice aged 8-12 wk and weighing 25-30 g purchased from Taconic Farms (Germantown, NY) were used for all experiments.

Animals in this study were maintained in accordance with the guidelines of the Committee on Animals of Harvard Medical School and those prepared by the Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council [Department of Health, Education, and Human Services, Publication No. 85-23 (National Institutes of Health), revised 1985].

Inhibition of complement activation in vivo. Mice that did not undergo aspiration injury were anesthetized with intraperitoneal pentobarbital sodium (60 mg/kg), and the alternative complement pathway was examined after injection with varying doses of sCR1 or sCR1sLex. The animals were killed with intraperitoneal pentobarbital sodium (90 mg/kg) 5 min after injection. Blood was aspirated from the right ventricle through a midline sternotomy and allowed to clot for 10 min at room temperature before being centrifuged at 4°C and 850 g for 10 min. The extracted serum was immediately frozen in dry ice and stored at -70°C. At a later date, the serum was defrosted and centrifuged at 2,500 g for 5 min before assay.

Alternative complement pathway hemolytic activity. The alternative complement pathway was examined using an assay specific to mouse complement hemolytic activity (3). Fresh citrated whole rabbit blood was washed three times with 0.9% saline and suspended in gelatin veronal buffer (Sigma), resulting in a concentration of 1.2 × 108 red blood cells/ml. Mouse sera were diluted in Mg2+-EGTA (7 mmol/l Mg2+ and 10 mmol/l EGTA). EGTA selectively chelates Ca2+, preventing activation of the classical pathway while allowing activation of the alternative pathway in the presence of Mg2+. Forty microliters of serially diluted serum were added to 25 µl of rabbit red blood cell solution and 25 µl of Mg2+-EGTA. The plate was shaken for 30 s and incubated for 1.5 h at 37°C. After centrifugation for 10 min at 850 g, 60 µl of each supernatant were collected. Absorbance was determined at 405 nm. To estimate the medium control and 100% lysis values, 65 µl of gelatin veronal buffer and deionized water were added to 25 µl of rabbit red blood cell solution, respectively.

Calculation. The lysis percentage was calculated as (sample OD - medium control OD)/(100% lysis OD - medium control OD). The data were plotted according to the transformation of the Von Krogh equation to determine the alternative complement pathway hemolytic activity titer per milliliter of undiluted mouse serum (11).

Aspiration protocol. Mice were anesthetized with intraperitoneal pentobarbital sodium and weighed, and their necks were washed with ethanol. Through a midline neck incision, a 0.75-cm-long, 16-gauge catheter (Angiocath; Becton Dickinson, Sandy, UT) was placed in the upper trachea under direct vision and secured with 4-0 silk sutures. The tracheostomy was cannulated with a 22-gauge, 1-cm-long Angiocath, and 2 ml/kg of 0.1 N HCl (Sigma, St. Louis, MO) or 0.9% saline (sham) were instilled in the trachea proximal to the carina using a 1-ml syringe. Previous studies performed in our laboratory have shown that this injury in mice is at maximum severity when 2 ml/kg of 0.1 N HCl were instilled (26). At that dose, experimental mortality is <10%. Before aspiration (5 min), 1 µCi of 125I-albumin (ICN, Irving, CA) in 0.3 ml of 0.9% saline was infused intravenously via a tail vein. Mice were allowed to breathe spontaneously. They were maintained in a supine position and kept anesthetized by intermittent intraperitoneal pentobarbital sodium injection. They were covered throughout the experiment to maintain body temperature.

After aspiration (4 h), the animals were killed by an intraperitoneal pentobarbital sodium overdose. Through a midline sternotomy, blood was aspirated from the right ventricle, and its gamma radioactivity was measured (Packard, Downers Grove, IL). Through the tracheostomy, the lungs were instilled with 0.5 ml of 0.9% saline using a 1-ml syringe. After 1 min, the BAL fluid was aspirated. This process was performed three times over a 3-min period using a total of 1.5 ml of 0.9% saline. A total of 0.9 ± 0.1 ml of bronchoalveolar lavage (BAL) fluid was retrieved. The lung permeability index (PI) is reported as the ratio of radioactivity per gram of BAL fluid retrieved to radioactivity per gram of blood.

An identical mouse preparation, save for the omission of administration of 125I-albumin, was used to quantitate neutrophils harvested from lung lavage. Another set of mice that underwent no lavage was used for immunohistochemical analysis of complement deposition. In this group of animals, the trachea and lungs were inflated with optimal cutting temperature compound (Miles) and immediately frozen.

Complement inhibition with sCR1 and sCR1sLex. Complement activation was inhibited by an intravenous bolus of sCR1 or sCR1sLex administered 5 min before acid aspiration. These agents were administered at predetermined doses shown to effectively inhibit complement activation in vivo. Another group of mice was treated with an intravenous bolus of sCR1 or sCR1sLex 0.5, 1, or 2 h after acid aspiration.

Neutrophil harvest from BAL fluid. Both lungs were lavaged five times over a period of 3 min using a total of 2.5 ml of saline with 0.6 mM EDTA. The lavage fluid return of 2.3 ± 0.1 ml was centrifuged at 1,500 rpm for 15 min. The cellular pellet was resuspended in 1 ml of cold saline. Fluid (200 µl) was injected in the cytospin apparatus (Shandon Lipshaw Cytospin 3) and was run at 200 rpm for 4 min. The glass slides were stained with Diff-Quik (Dade, Düdingen, Switzerland) to identify macrophages. PMN counts were performed in a blinded fashion. The results are expressed as total PMN count in BAL fluid (means ± SE).

Immunohistological analysis. A group of mice was killed with intraperitoneal pentobarbital sodium 0.5 and 1 h after acid aspiration, and the lungs were harvested. During this period, endothelial P-selectin is thought to be upregulated significantly (19). Immunohistological localization of sCR1 was performed on cryostat sections of snap-frozen lung after paraformaldehyde-methanol fixation using a peroxidaseanti-peroxidase method as previously described (12). The primary antibody used was rabbit polyclonal IgG antibody to human sCR1 (AVANT Immunotherapeutics) at 0.5 µg/ml. Localization was visualized with the chromogen diaminobenzidine, and slides were counterstained with hematoxylin for microscopy.

Statistical analysis. Results are presented as means ± SE in the text and Figs. 1-4. Groups were subjected to one-way ANOVA, and when significance was found, Student's t-test with the Bonferroni correction for multiple comparisons was applied. Percentage reduction in PI was calculated after subtraction of the background value determined in sham animals.


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Fig. 1.   Dose response of soluble complement receptor type 1 (sCR1) and sialyl Lewisx-glycosylated sCR1 (sCR1sLex) in inhibiting the in vivo alternative complement pathway hemolytic activity (APCH50) of mouse serum. sCR1 and sCR1sLex demonstrated a 9% difference in their ability to inhibit complement activation when given at 1 mg/kg. At doses of 5 and 10 mg/kg, both agents were similar in their capacity to inhibit complement activation (n = 6/time point). *P < 0.05.



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Fig. 2.   Comparison of sCR1 and sCR1sLex in moderating acid aspiration injury. All groups underwent acid aspiration. The respective permeability indexes after treatment with 5 mg/kg sCR1 and 5 mg/kg sCR1sLex were 44 and 67% lower compared with those in the untreated group. Treatment with 10 mg/kg sCR1sLex reduced permeability by 82%, significantly more than the 54% reduction noted in animals treated with 10 mg/kg sCR1. *P < 0.05 compared with untreated mice. dagger P < 0.05 compared with sCR1-treated group.



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Fig. 3.   Role of delayed treatment with sCR1 and sCR1sLex after acid aspiration. All groups underwent acid aspiration. The respective permeability indexes after treatment with 10 mg/kg sCR1 at 0.5 and 1 h after acid aspiration were 45 and 38%, significantly less than that in the untreated group. The permeability index after treatment with 10 mg/kg sCR1sLex 0.5 and 1 h after acid aspiration was moderated by 70 and 57%, respectively, significantly less than that in the sCR1-treated groups. No significant reduction in permeability was observed after treatment 2 h after acid aspiration for either agent. *P < 0.05 compared with injured untreated mice. dagger P < 0.05 compared with sCR1-treated group.



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Fig. 4.   Immunostaining for sCR1 in mouse lung after 0.5 h of acid aspiration. A: sCR1sLex; B: sCR1. The 1-h immunohistochemistry is identical to 0.5 h and therefore is not shown. sCR1 was localized on the vascular endothelium of sCR1sLex-treated mice but not in animals treated with sCR1.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Inhibition of complement activation in vivo. Having established the presence of sLex tetrasaccharide in the Lec11 glycoproteins as described above, it was important to examine the effects of such glycosylation on complement inhibitory activation. At 1 mg/kg, sCR1 was marginally more effective than sCR1sLex in inhibiting complement activation, indicating a possible effect of differing glycosylation in these assays (21). At doses of 5 and 10 mg/kg, sCR1 and sCR1sLex exhibited very similar complement inhibitory properties, with both agents providing >99% complement inhibition at 10 mg/kg (Fig. 1).

Moderation of injury by sCR1sLex. Acid aspiration in untreated mice (n = 26) led to a marked rise in PI of 0.100 ± 0.007 compared with that in saline-aspirated sham animals (n = 10), which had a PI of 0.007 ± 0.001 (P < 0.05). PI was 44% lower in mice treated with 5 mg/kg sCR1 (n = 20, PI 0.059 ± 0.003, P < 0.05) and 54% lower in 10 mg/kg sCR1-treated animals (n = 17, PI 0.050 ± 0.004, P < 0.05). Respective PI reductions observed in mice treated with 5 and 10 mg/kg sCR1sLex of 0.038 ± 0.004 (67%, n = 20) and 0.024 ± 0.002 (82%, n = 19) were greater compared with those in the respective sCR1-treated groups (P < 0.05; Fig. 2).

Effects of delayed treatment after acid aspiration. Respective PI reductions observed in mice treated 0.5 and 1 h after acid aspiration with 10 mg/kg sCR1sLex of 0.035 ± 0.003 (70%, n = 19) and 0.045 ± 0.004 (57%, n = 20) were greater than the respective decreases in PI of 0.058 ± 0.005 (45%, n = 20) and 0.065 ± 0.005 (38%, n = 19) observed in the 10 mg/kg sCR1-treated groups (P < 0.05). Vascular permeability was not significantly moderated after treatment with sCR1sLex (n = 15) or sCR1 (n = 16) 2 h after acid aspiration (PI of 0.102 ± 0.008 and 0.097 ± 0.008, respectively; Fig. 3).

Role of sLex in PMN adhesion. To test whether sCR1sLex moderated lung permeability by antagonism of neutrophil-endothelial selectin adhesion in addition to complement inhibition, a group of animals had their lungs lavaged, and the cells were counted. Acid aspiration produced an accumulation of 479 ± 132 PMNs in untreated mice (n = 5), similar to 506 ± 148 PMNs in animals treated with 10 mg/kg sCR1 (n = 5). As expected, the counts for saline-aspirated mice were significantly lower. Treatment with 10 mg/kg sCR1sLex before acid aspiration significantly reduced PMN counts in the BAL fluid. Delayed treatment 0.5 and 1 h after acid aspiration was also effective. There was no significant reduction in PMN counts after treatment with sCR1sLex 2 h after acid aspiration (Table 1).

                              
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Table 1.   PMN accumulation in bronchoalveolar fluid

Immunohistological analysis. Lung samples snap-frozen in optimal cutting temperature compound 0.5 and 1 h after acid aspiration were stained for human sCR1. sCR1 was localized on the vascular endothelium of sCR1sLex-treated mice but not in animals treated with sCR1 (Fig. 4).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The dependence of acid aspiration lung injury on complement activation has been reported previously and is confirmed in these studies with a 54% reduction in permeability in mice treated with 10 mg/kg sCR1 (13, 26). In general, complement activation can induce local injury by neutrophil chemoactivation and directly via the membrane attack complex (16, 17). This proinflammatory role of C5b-9 has been documented recently in our mouse model of acid aspiration injury (13). In that study, C5-deficient animals unable to form the C5b-9 membrane attack complex were protected from injury to the same degree as wild-type animals treated with the complement antagonist sCR1. In addition, the injury was restored in C5-deficient mice reconstituted with wild-type serum. These data suggested that, with regard to the complement segment of the inflammatory reaction, the terminal complement components are the key mechanism by which the alternative pathway exerts its effect. Furthermore, to examine the interaction of complement and neutrophils, particularly with regard to the anaphylatoxin C5a, a group of C5-deficient animals was immunodepleted of neutrophils before undergoing aspiration. This led to a significant reduction in injury compared with neutrophil-replete C5-deficient mice, indicating a role for the neutrophil in this injury without C5, C5a, or other terminal complement products (13). Further evidence that C5a was not operative in this setting as a chemoattractant was provided by the observation that neutrophil accumulation in BAL fluid was similar in both injured wild-type and C5-deficient animals. Thus these data strongly suggested to us that the complement action in mediating injury was via formation of the membrane attack complex (13). In addition, in that same study, our observation of a 90% reduction in permeability after combined PMN depletion and complement inhibition with sCR1, representing 30% more protection than just complement inhibition, further supported the concept of a neutrophil-mediated injury independent of complement activation (13). Thus the combined action of an agent that could offer both complement and neutrophil antagonism would be anticipated to be more effective than complement inhibition alone (8, 13).

Previous experimental work in acid aspiration has shown that anti-beta 2-integrin therapy in the form of immunoneutralization of CD18 did not affect either PMN sequestration or lung injury (6, 7). We therefore speculated that the endothelial selectins are the operative adhesion molecules after acid aspiration. Support for this is provided by our recent observations in mice treated with recombinant PSGL-Ig fusion protein, a known P- and E-selectin antagonist in which injury was moderated to the same extent as after PMN immunodepletion (14).

In the present study, acid-aspirated mice treated with 10 mg/kg sCR1sLex demonstrated an 82% reduction in permeability, representing 28% more protection from injury than animals treated with 10 mg/kg sCR1. This increased efficacy of sCR1sLex compared with sCR1 in protecting against acid aspiration injury may be because of its binding of selectin adhesion molecules on the activated vascular endothelium, as indicted by immunohistochemistry. This is consistent with the significant moderation of PMN sequestration in the BAL fluid in animals treated with sCR1sLex in contrast to sCR1. This indicates that sLex inhibits neutrophil-endothelial selectin adhesion in acid aspiration. Further supporting evidence is provided by in vitro studies that demonstrate that sCR1sLex but not sCR1 bind cell surface E-selectin- and block P-selectin-mediated cellular adhesion (21). In addition to the possibility that sCR1sLex may compete with the ability of neutrophils to bind to their selectin counterreceptors on endothelial cells, this hybrid molecule may also allow for complement inhibition to be focused at the site of the activated endothelium (18).

Because treatment invariably takes place after the aspiration event, the ability of complement inhibition and endothelial selectin blockade after acid aspiration to moderate injury was studied. Progressive delay in application of complement inhibition with sCR1 was associated with progressive increases in permeability, although significant protection of 45 and 38% was observed with treatment 0.5 and 1 h after acid aspiration compared with that in untreated groups. The therapeutic effects of sCR1 were absent when delivery was given 2 h after acid aspiration. Delayed treatment with sCR1sLex 0.5 and 1 h after acid aspiration provided 70 and 57% reduction in permeability, respectively, significantly better than treatment with sCR1. This indicates that there was continued benefit of complement inhibition, possibly relating to a delay between acid aspiration and systemic complement activation. That there was also a benefit of delayed antagonism of PMN-endothelial selectin adhesion was shown by the significant decrease in neutrophil accumulation in the alveolar spaces. It is likely that neutrophil adhesion mechanisms are endothelial selectin dependent up to that time point (14). These data indicate that there is a therapeutic window of up to 1 h after acid aspiration where injury can be moderated by both complement and neutrophil antagonism. As with sCR1, infusion of sCR1sLex 2 h after acid aspiration failed to significantly moderate injury compared with that in the untreated group.

In conclusion, our data indicate that, in experimental acid aspiration, sCR1sLex localizes on the vascular endothelium and effectively moderates permeability by antagonizing both complement activation and neutrophil adhesion. These data also support our previous observations of neutrophil-mediated injury in a complement-independent fashion. In addition, PMN-endothelial selectin interaction is an important mechanism of neutrophil-mediated injury. Finally, delayed therapy with complement and neutrophil antagonists may prove to be beneficial after acid aspiration.


    ACKNOWLEDGEMENTS

We thank Dr. Henry Marsh, Jr., and Dr. Carolyne Petty of AVANT Immunotherapeutics, Needham, MA, for the generous donation of sCR1, sCR1sLex, and anti-sCR1 antibody used in the experiments and Amy C. Imrich for performing the immunohistochemistry.


    FOOTNOTES

This work was supported in part by National Institute of General Medical Sciences Grants GM-35141, GM-24891, and GM-52585, The Brigham Surgical Group, and The Trauma Research Foundation.

Address for reprint requests and other correspondence: H. B. Hechtman, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115 (E-mail: HHechtman{at}Partners.org).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 26 March 2001; accepted in final form 28 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Lung Cell Mol Physiol 281(6):L1494-L1499
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