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Effects of recombinant surfactant protein C and elastase inhibitor on neutrophil apoptosis

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with mortality in excess of 50%. About 5–35% of patients with sepsis develop ARDS and the condition is characterised by infiltration and accumulation of activated polymorphonuclear leukocytes (PMNL) within the capillary and alveolar spaces. Increased alveolar neutrophil elastase and decreased antiprotease activity also occurs. Elastase directly damages surfactant-specific protein in vitro and alters surfactant function. Recombinant surfactant protein C (r-SPC) improves pulmonary dynamics, oxygenation and reduces hyaline membrane formation in animal models of ALI/ARDS. Accumulated neutrophils are removed by programmed cell death or apoptosis, resulting in resolution and healing. We determined the effect of r-SPC with and without elastase inhibitor (EI) on neutrophil apoptosis.

Heparinised venous blood samples were obtained from 10 healthy adult volunteers, and PMNL were isolated by density gradient centrifugation and suspended in minimum essential medium supplemented with 10% autologous plasma. Cells were incubated for 16 hours with either 2 μg/ml lipopolysaccharide (LPS) (stimulated) or phosphate-buffered saline (unstimulated) in the presence of r-SPC or EI, or both. Cells were washed and treated with Annexin V/propidium iodide stains and analysed by dual-laser flow cytometry. The percentage of early apoptotic cells was determined using four-quadrant analysis. Data were analysed using Friedman analysis of variance with post hoc testing by Wilcoxon signed ranks testing for paired data (Table 1).

Table 1 Table 1

The results of this pilot study show that r-SPC significantly increases activated PMNL apoptosis, and this is maintained in the presence of EI. This suggests the possible use of r-SPC and elastase inhibitor in inducing neutrophil apoptosis and hence clearance of neutrophils in ALI/ARDS. Further studies are needed.

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Keywords

  • Acute Lung Injury
  • Acute Respiratory Distress Syndrome
  • Density Gradient Centrifugation
  • Polymorphonuclear Leukocyte
  • Neutrophil Elastase