Relationship of proteinases and proteinase inhibitors with microbial presence in chronic lung disease of prematurity

Abstract

Background: A proteolytic imbalance has been implicated in the development of "classical" chronic lung disease of prematurity (CLD). However, in "new" CLD this pattern has changed. This study examines the longitudinal relationship between neutrophil proteinases and their inhibitors in ventilated preterm infants and their relationship to microbial colonisation.

Methods: Serial bronchoalveolar lavage fluid was obtained from ventilated newborn preterm infants. Neutrophil elastase (NE) activity, cell counts, metalloproteinase (MMP)-9, MMP-9/TIMP-1 complex, SerpinB1 concentration and percentage of SerpinB1 and alpha(1)-antitrypsin (AAT) in complex with elastase were measured. The presence of microbial genes was examined using PCR for 16S rRNA genes.

Results: Statistically more infants who developed CLD had NE activity in at least one sample (10/20) compared with infants with resolved respiratory distress syndrome (RDS) (2/17). However, NE activity was present in a minority of samples, occurring as episodic peaks. Peak levels of MMP-9, MMP-9/TIMP-1 complex, percentage of AAT and SerpinB1 in complex and cell counts were all statistically greater in infants developing CLD than in infants with resolved RDS. Peak values frequently occurred as episodic spikes and strong temporal relationships were noted between all markers. The peak values for all variables were significantly correlated to each other. The presence of bacterial 16S rRNA genes was associated with the development of CLD and with elevated elastase and MMP-9.

Conclusion: NE activity and MMP-9 appear to be important in the development of "new" CLD with both proteinase and inhibitor concentrations increasing episodically, possibly in response to postnatal infection.

Figure 1

Longitudinal analysis of bronchoalveolar lavage fluid from three infants showing temporal relationships between variables. Graphs A–C show SerpinB1 concentrations (left axis, solid line) and the proportion of α₁-antitrypsin (AAT) in complex with serine protease (right axis, broken line) relative to day of life (x-axis). Graphs D–F show the same infants with matrix metalloproteinase (MMP)-9 concentrations (left axis, solid line) and total cell counts (right axis, broken line) against days of life. The arrows indicate the days when free neutrophil elastase (NE) was detected. No free NE was detected in infant 3.

Figure 2

Western blot of α₁-antitrypsin (AAT) species in sequential bronchoalveolar lavage (BAL) fluid specimens for (A) a representative infant from the resolved respiratory distress syndrome group in whom active neutrophil elastase (NE) was not detected and (B) an infant from the chronic lung disease of prematurity group in whom active NE was detected. Patient age and NE concentrations are shown below each lane of the western blot. Equal amounts of protein were loaded for analysis and the left lanes show purified plasma AAT migrating at 53 kDa (standard). Additional bands in BAL fluid specimens are the complex with serine proteases at 80 kDa and degraded AAT at 48 kDa. Note that the day 1 samples for both infants contain free AAT only, while day 5 BAL fluid for patient A and days 2–6 for patient B also contain an additional higher AAT-proteinase complex. BAL fluid from days 2–6 of patient B also contains degraded AAT.

Figure 3

Western blot of SerpinB1 species in sequential bronchoalveolar lavage (BAL) fluid specimens of an infant with chronic lung disease of prematurity in whom active neutrophil elastase (NE) was detected. Equal volumes of BAL fluid were loaded for analysis and the left lane shows purified SerpinB1 (10 ng) migrating at 42 kDa (standard). Other species present in BAL fluid are the 66 kDa complex with serine proteinase, a partially degraded intermediate product and degraded SerpinB1 at 38 kDa. A statistically significant increase in both the total concentration and percentage in the complex is seen, occurring with increased NE activity.

Figure 4

Scatterplots of (A) peak elastase activity and (B) matrix metalloproteinase (MMP)-9 concentrations for each diagnostic class of infant. Filled symbols indicate 16S rRNA gene-positive specimens and empty boxes indicate negative specimens. Sequencing data for 16S rRNA gene-positive organisms are also shown. Some samples were identified as 16S rRNA gene-positive but no specific microorganism was identified by sequencing. Abbreviations for micro-organisms include S epi (S epidermidis), S haem (S haemolyticus) and P aeru (P aeruginosa). CLD, chronic lung disease of prematurity; RDS, respiratory distress syndrome.