Protective effects of sialyl Lewis X and anti-P-selectin antibody against lipopolysaccharide-induced acute lung injury in rabbits

Abstract

The prophylactic effects of selectin inhibitors on lipopolysaccharide-induced acute lung injury were studied in rabbits by using sialyl Lewis X-oligosaccharide and PB1.3, an anti-human P-selectin monoclonal antibody. Lipopolysaccharide-induced acute lung injury resembles that of the acute respiratory distress syndrome, in which there is a decrease in arterial blood oxygen tension (PaO₂) and an increase in the difference between alveolar and arterial oxygen tension (A-aDO₂). Prophylactic treatment with the selectin inhibitors, sialyl Lewis X-oligosaccharide (55 mg kg⁻¹ i.v. bolus injection immediately before lipopolysaccharide administration+36 mg kg⁻¹ h⁻¹ i.v. infusion for 4 h) and PB1.3 (5 mg kg⁻¹ i.v. bolus injection immediately before lipopolysaccharide administration), prevented the lipopolysaccharide-induced impairments in pulmonary gas exchange. In contrast, these agents had no significant effects on lipopolysaccharide-induced systemic hypotension, the decrease in the number of circulating white blood cells and platelets, the decline in blood pH, or the increase in arterial CO₂ tension (PaCO₂). These results indicate that selectin inhibitors including sialyl Lewis X-oligosaccharide and the anti-P-selectin antibody, PB1.3, attenuate lipopolysaccharide-induced acute lung injury in rabbits. This is the first demonstration that P-selectin is directly involved in the development of lipopolysaccharide-induced impairments in pulmonary gas exchange.

Introduction

Acute respiratory distress syndrome is an acute, progressive pulmonary disorder characterized by inflammation and increased permeability pulmonary edema, and is associated with hypoxemia, increased lung compliance, and diffuse pulmonary infiltrates on chest radiography (Tate and Repine, 1983). Septic shock caused by Gram-negative bacteria is thought to be one of the most common conditions associated with acute lung injury, including acute respiratory distress syndrome. Acute respiratory distress syndrome develops in approximately 70–80% of patients with Gram-negative sepsis and is associated with a high mortality rate (Sachdeva and Guntupalli, 1997; Artigas et al., 1998). In the last 10 years, a number of novel therapeutic approaches, including the use of anti-cytokine (Natanson et al., 1994), surfactant (Gregory et al., 1997) and anti-oxidant agents (Bernard et al., 1997), have been developed and tested against acute respiratory distress syndrome with sepsis, but few have proved beneficial in reducing mortality. The lipopolysaccharide-induced experimental lung injury model is associated with systemic hypotension, impairment of pulmonary oxygenation, coagulation abnormalities and acidosis in several animal species (Granger and Kubes, 1994; Carvalho et al., 1997). These models have been used to investigate various agents including lysofylline, an inhibitor of phosphatidic acid generation (Hasegawa et al., 1997), methylpredonisolone (Borg et al., 1985), a neutrophil elastase inhibitor (Nishina et al., 1997), nafamostat mesilate (Uchiba et al., 1997) and an anti-interleukin-8 antibody (Carvalho et al., 1997).

The initial step in the acute inflammatory processes, including that of acute respiratory distress syndrome, is the adherence of polymorphonuclear leukocytes to activated endothelial cells (Granger and Kubes, 1994). This first contact and the subsequent rolling step depend largely on the interaction between selectins (E-, L-, and P-selectins) and their ligands including sialyl Lewis X-oligosaccharide (Bevilacqua and Nelson, 1993). The polymorphonuclear leukocytes that adhere and migrate to sites of inflammation undergo functional activation and release various mediators, including reactive oxygen radicals, proteolytic enzymes and possibly inflammatory cytokines, which subsequently cause severe damage to the underlying endothelial cells.

Three selectin family members are glycoproteins that share structurally homologous domains, namely, the lectin, epidermal growth factor, complement binding, and cytoplasmic domains (Bevilacqua and Nelson, 1993). L-selectin is constitutively expressed on leukocytes and is rapidly shed following leukocyte activation. The expression of E-selectin is induced by inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β and lipopolysaccharide via de novo protein synthesis. In contrast, P-selectin is stored in α-granules of platelets and in Weibel–Palade bodies of endothelial cells and is rapidly expressed on the cell surface in response to various stimuli such as thrombin and histamine. The sialyl Lewis X-oligosaccharide motif is a carbohydrate ligand for all three selectins (Bevilacqua and Nelson, 1993) and sialyl Lewis X-oligosaccharide has been used as a selectin inhibitor in various studies. Sialyl Lewis X-oligosaccharide inhibits selectin-mediated polymorphonuclear leukocyte adhesion in vitro (Foxall et al., 1992; Kawamura et al., 1995) and attenuates ischemia/reperfusion-induced myocardial necrosis in canine (Lefer et al., 1994), rabbit (Yamada et al., 1998), and rat (Tojo et al., 1996) models as well as ischemia/reperfusion injury in the rabbit ear (Han et al., 1995). We have recently observed that sialyl Lewis X-oligosaccharide attenuates neutrophil-dependent myocardial dysfunction in the isolated rat heart (Ohnishi et al., 1999).

Selectin inhibitors have also been used in various lung injury models, primarily in small animals such as the rat. For example, in rats given lipopolysaccharide intratracheally, the accumulation of polymorphonuclear leukocyte in bronchoalveolar lavage fluid was found to be reduced by the intravenous administration of anti-E-selectin mAb and soluble E-selectin, respectively (Ulich et al., 1994). In the immune-complex-induced lung injury model in rats, the administration of sialyl Lewis X-oligosaccharide (Mulligan et al., 1993) and an anti-P-selectin antibody (Bless et al., 1998) resulted in protection against the injury. In these lung injury models, the effects of drugs are evaluated primarily in terms of inflammatory parameters such as permeability, hemorrhage and myeloperoxidase. The effect of the drugs cannot be evaluated in terms of functional parameters of the lung, such as gas exchange, partly due to the technical difficulties associated with the small animal species used. In contrast, pulmonary function can be analyzed in larger animals, such as pig and rabbit.

Recently, Ridings et al. (Ridings et al., 1997) showed that sialyl Lewis X-oligosaccharide protected against the pulmonary dysfunction induced by the infusion of live Pseudomonas aeruginosa in a porcine sepsis model. They observed that the administration of sialyl Lewis X-oligosaccharide improved arterial oxygenation and various inflammatory parameters, thus, demonstrating that the inhibition of selectins protects against lung injury. Since sialyl Lewis X-oligosaccharide inhibits all three selectins (Foxall et al., 1992), their results implied that the inhibition of all three selectins may be protective. However, the possible role of each selectin molecule could not be elucidated from their findings.

We have recently demonstrated that the systemic administration of lipopolysaccharide results in an increase in the plasma P-selectin level in rats (Misugi et al., 1998). Based on this finding and the observations that patients with acute lung injury (Sakamaki et al., 1995) and sepsis (Fijnheer et al., 1997) show an elevated level of soluble P-selectin, we hypothesize that P-selectin is closely involved in the development of pulmonary dysfunction. No study, however, has demonstrated the specific role of P-selectin in the development of pulmonary dysfunction. In the present study, we investigated whether selectin inhibitors can protect against pulmonary dysfunction in a rabbit model by using sialyl Lewis X-oligosaccharide, and then demonstrated the specific contribution of P-selectin by using PB1.3, an anti-P-selectin monoclonal antibody.