Surfactant protein-d inhibits lung inflammation caused by ventilation in premature newborn lambs

Abstract

Rationale: Premature newborns frequently require manual ventilation for resuscitation during which lung injury occurs. Although surfactant protein (SP)-D regulates pulmonary inflammation, SP-D levels are low in the preterm lung. Commercial surfactants for treatment of respiratory distress syndrome do not contain SP-D.

Objectives: To determine whether addition of recombinant human SP-D (rhSP-D) to commercial surfactant influences lung inflammation in ventilated premature newborn lambs.

Methods: Prematurely delivered lambs (130 d gestation age) were resuscitated with 100% O(2) and peak inspiratory pressure 40 cm H(2)O for 20 minutes and then treated with Survanta or Survanta containing rhSP-D. Ventilation was then changed to regulate tidal volume at 8 to 9 ml/kg. At 5 hours of age lambs were killed for sample collection.

Measurements and main results: Sequential blood gas and tidal volume were similar in lambs treated with or without rhSP-D, indicating that lung immaturity and ventilatory stress used to support premature lambs were comparable between the two groups. Ventilation caused pulmonary inflammation in lambs treated with surfactant alone. In contrast, surfactant containing rhSP-D decreased neutrophil numbers in bronchoalveolar lavage fluid and decreased neutrophil elastase activity in lung tissue. IL-8 mRNA and IL-8 protein were significantly decreased in the +rhSP-D group lamb lungs, to 20% of those in controls. The addition of rhSP-D also rendered Survanta more resistant to plasma protein inhibition of surfactant function.

Conclusions: Treatment with rhSP-D-containing surfactant inhibited lung inflammation and enhanced the resistance of surfactant to inhibition, supporting its potential usefulness for prevention of lung injury in the preterm newborn.

Figures

Figure 1.

Lung physiology. Per protocol, ventilation was carefully regulated for both groups. (A) Premature newborn lambs were resuscitated after birth by ventilation with peak inspiratory pressure (PIP) 40 cm H2O, resulting in (B) mean Pco2 40 mm Hg and (C) mean Vt 11 ml/kg for both groups. Surfactant was given at 20 minutes of age and ventilation was changed (C) to regulate Vt at 8 to 9 ml/kg, requiring (A) a mean PIP of 27 cm H2O for both groups. These data indicate that lung immaturity, as well as ventilatory support of the premature lambs, was comparable between control lambs and lambs treated with recombinant human SP-D (+rhSP-D). Some error bars are within symbols.

Figure 2.

Modified ventilation index (MVI) and Po2/Fio2. (A) MVI was calculated as peak inspiratory pressure × Pco2 × respiratory rate/1,000. Although not significant, MVI tends to be better (lower) for the group treated with recombinant human surfactant protein D (+rhSP-D) group at later times. (B) Po2/Fio2 was higher in +rhSP-D group compared with control. *P < 0.01 by two-way repeated measures analysis of variance (ANOVA) (overall comparison of control versus +rhSP-D group). Po2/Fio2 was significantly decreased with time after 210 minutes in control group (P < 0.05 vs. 18 min by one-way ANOVA).

Figure 3.

Pressure-volume curves and lung histology. (A) The deflation limbs of pressure-volume curves were not different between the groups. (B, C) Lung histology assessed after staining with hematoxylin and eosin was similar for both groups. Histology was typical of immature lung, including thickened alveolar septal walls and patchy atelectasis. More alveolar fluid was observed in control lambs than in lambs treated with recombinant human surfactant protein D (+rhSP-D). Scale bar: 100 μm.

Figure 4.

Inflammatory cells in bronchoalveolar lavage fluid (BALF) and neutrophil elastase (NE) activity in lung homogenates. (A) Increased total inflammatory cells and neutrophils in BALF induced by ventilation were suppressed by recombinant human surfactant protein D (rhSP-D). (B) NE activity was assessed by a spectrophotometric assay using a chromogenic substrate specific for NE as described in Methods. Treatment with rhSP-D–containing Survanta decreased NE activity. *P < 0.05 versus control.

Figure 5.

Proinflammatory cytokines, keratinocyte-derived chemokine (KC), and monocyte chemotactic protein 1 (MCP1) in lung homogenates. (A) Increased expression of IL-8 mRNA was significantly suppressed by recombinant human surfactant protein D (rhSP-D) treatment. Although not statistically significant due to the large variation in the control group, mean values of IL-6, IL-1β, KC, and MCP1 were generally lower in the +rhSP-D group. (B) IL-8 protein in lung homogenates was significantly decreased by rhSP-D treatment. IL-1β was not influenced by rhSP-D treatment. *P < 0.05 versus control.

Figure 6.

Resistance to surfactant inhibition and effects on the ultrastructure of Survanta. (A) Surface tension was measured by a captive bubble surfactometer. Survanta (+ buffer) had high surface activity and minimum surface tension was low and was not influenced by addition of recombinant human surfactant protein D (rhSP-D). Plasma protein inhibited surface tension–lowering properties of Survanta and minimum surface tension was increased. The addition of rhSP-D rendered the Survanta more resistant to plasma protein inhibition. Minimum surface tension was low in the presence of plasma protein. n = 3, *P < 0.05 versus Survanta + buffer with plasma. (B, C) Representative electron micrographs of Survanta mixed with buffer or rhSP-D. Addition of rhSP-D changed ultrastructure of Survanta from simple lipid layers to the mixture of multiple lipid layers and lipid aggregates. n = 3 per group. Scale bar: 500 nm.