Role of serine proteases in the regulation of interleukin-877 during the development of bronchopulmonary dysplasia in preterm ventilated infants.

RATIONALE: The chemokine interleukin-8 is implicated in the development of bronchopulmonary dysplasia in preterm infants. The 77-amino acid isoform of interleukin-8 (interleukin-877) is a less potent chemoattractant than other shorter isoforms. Although interleukin-877 is abundant in the preterm circulation, its regulation in the preterm lung is unknown. OBJECTIVES: To study expression and processing of pulmonary interleukin-877 in preterm infants who did and did not develop bronchopulmonary dysplasia. METHODS: Total interleukin-8 and interleukin-877 were measured in bronchoalveolar lavage fluid from preterm infants by immunoassay. Neutrophil serine proteases were used to assess processing. Neutrophil chemotaxis assays and degranulation of neutrophil matrix metalloproteinase-9 were used to assess interleukin-8 function. MAIN RESULTS: Peak total interleukin-8 and interleukin-877 concentrations were increased in infants who developed bronchopulmonary dysplasia compared to those who did not. Shorter forms of interleukin-8 predominated in the preterm lung (96.3% No-bronchopulmonary dysplasia vs 97.1% bronchopulmonary dysplasia, p>0.05). Preterm bronchoalveolar lavage fluid significantly converted exogenously added interleukin-877 to shorter isoforms (p<0.001). Conversion was greater in bronchopulmonary dysplasia infants (p<0.05). This conversion was inhibited by α-1 antitrypsin and antithrombin III (p<0.01). Purified neutrophil serine proteases efficiently converted interleukin-877 to shorter isoforms in a time- and dose-dependent fashion; shorter interleukin-8 isoforms were primarily responsible for neutrophil chemotaxis (p<0.001). Conversion by proteinase-3 resulted in significantly increased interleukin-8 activity in vitro (p<0.01). CONCLUSIONS: Shorter, potent, isoforms interleukin-8 predominate in the preterm lung, and are increased in infants developing bronchopulmonary dysplasia, due to conversion of interleukin-877 by neutrophil serine proteases and thrombin. Processing of interleukin-8 provides an attractive therapeutic target to prevent development of bronchopulmonary dysplasia.

Role of interleukin-6, its receptor and soluble gp130 in chronic lung disease of prematurity.

BACKGROUND: Interleukin-6 (IL-6) signalling involves the interplay between IL-6, soluble IL-6 receptor (sIL-6R) and soluble gp130 (sgp130). IL-6 activity is modulated by the soluble receptors to produce both pro- and anti-inflammatory effects in human diseases and animal models. The expression and functional activity of these molecules in lungs of preterm ventilated infants is unknown. OBJECTIVES: We investigated this pathway in preterm infants who were at risk of developing chronic lung disease of prematurity (CLD). METHODS: Cytokines and soluble receptors were measured in bronchoalveolar lavage fluid (BALF) from ventilated preterm infants ≤32 weeks of gestation who did or did not develop CLD. B9 cells, which specifically proliferate to IL-6, were used to assess BALF IL-6 functional activity. RESULTS: Inflammatory cells, IL-8 and monocyte chemotactic protein-1 were increased in BALF from the CLD group when compared to the No CLD group (p < 0.05). BALF IL-6 and sIL-6R were similar in both groups. In contrast, BALF sgp130 and sgp130/sIL-6R were greater in the CLD group when compared to the No CLD group (p = 0.01 and p = 0.02, respectively). However, the increased BALF sgp130 did not appear to modulate the BALF IL-6 functional activity. CONCLUSION: Lung inflammation was observed in the CLD group. Increased BALF sgp130 was noted in the CLD group but it did not appear to modulate the pulmonary IL-6 bioactivity. Further research is needed to investigate the potential modulatory activity of sgp130 in the preterm lung.

Increased prevalence of low oligomeric state surfactant protein D with restricted lectin activity in bronchoalveolar lavage fluid from preterm infants.

BACKGROUND: Surfactant protein D (SP-D) is a soluble oligomeric C-type lectin known to protect against lipopolysaccharide and ventilator-induced lung injury in preterm lambs. Here we assess the expression and functional status of SP-D in bronchoalveolar lavage fluid (BALF) from preterm infants at risk of chronic lung disease (CLD) of prematurity and term controls. This is the first systematic evaluation of SP-D function in any clinical cohort. METHODS: SP-D was quantified in BALF from 28 ventilated preterm infants and five ventilated term infants. SP-D lectin activity was tested in a zymosan binding assay followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot in BALF from the same infants. SP-D lectin activity was also tested towards maltose-agarose and mannan for selected BALF samples. RESULTS: SP-D expression was lower on day 1 in those preterm infants who subsequently developed CLD but increased over the first 5 days of life in term and preterm neonates. The percentage of neonatal SP-D capable of binding zymosan rarely exceeded 50% in any BALF sample and was 3.5 times lower in preterm infants than term infants on day 1 of life. Similar binding defects were observed towards maltose-agarose and mannan. SDS-PAGE analysis revealed that zymosan-bound SP-D was more highly oligomerised (≥12-mers) than unbound SP-D, which was composed primarily of trimers and lower oligomeric forms. CONCLUSIONS: Substantial and functionally relevant variation in the expression and oligomeric distribution of SP-D exists between preterm and term neonatal lung secretions. This has implications for proposed SP-D replacement therapy in this population.

Inactivation of IL-6 and soluble IL-6 receptor by neutrophil derived serine proteases in cystic fibrosis.

The ability of IL-6 to signal via both membrane bound and soluble receptors is thought to explain the capacity of this cytokine to act in both the initiation and resolution of acute inflammatory responses. In cystic fibrosis (CF), poorly resolved neutrophillic inflammation of the lungs is a primary cause of morbidity and mortality. Expression of IL-6 has been reported to be low in CF lung secretions, despite ongoing inflammation, but the status of soluble IL-6 receptor (sIL-6R) in these patients is unknown. We hypothesised that sIL-6R may be an important potentiator of IL-6 activity in CF associated lung disease. IL-6, sIL-6R and sgp130 (a natural antagonist of responses mediated by the sIL-6R) were analysed by ELISA and Western blot in bronchoalveolar lavage fluid (BALF) from 28 paediatric CF patients and nine non-CF controls. Total cell counts in CF were four fold higher compared to controls (median: 1.4 x 10(6) cells/ml v. 0.35 x 10(6) cells/ml in controls) (p<0.001) and the infiltrate was dominated by neutrophils which were elevated by 89 fold (0.62 x 10(6) cells/ml v. 0.007 x 10(6) cells/ml in controls) (p<0.001). Other markers of inflammation such as IL-8 and MCP-1 were elevated 17.5 and 3.8 fold respectively (IL-8; median: 1122 pg/ml v. 64 pg/ml in controls, p<0.01 and MCP-1; median: 692 pg/ml v. 182 pg/ml in controls, p<0.05). IL-6, although present in 23/32 CF BALF specimens compared to 1/9 controls (p<0.01), was weakly expressed (median: 50 pg/ml). Expression of sIL-6R and sgp130 in CF was no different to control patients. We tested whether weak expression of all three molecules was due to degradation by CF BALF. Degradative activity was observed in association with BALF elastase activity and could be specifically blocked by serine protease inhibitors. Degradation of sIL-6R by purified serine proteases (elastase, cathepsin G and proteinase 3) was also observed leading to a loss of trans-signalling activity. Interestingly, sIL-6R was protected from proteolysis by interaction with IL-6. Our data identify and define a novel protease mediated deficiency of IL-6 signalling in the CF lung.

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

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.

Monoclonal anti-neutrophil elastase antibody characterisation: ability to block function, detect free versus serpin-complexed enzyme and stain intracellular granules.

Four commercially available monoclonal antibodies (clones NP57, 256-3K1, 39A and 203) were characterised for their ability to block human neutrophil elastase (HNE) activity; capture free purified HNE or neutralised HNE in complex with alpha-1-antitrypsin (AAT); detect HNE and HNE-AAT by Western blot analysis; and detect intracellular HNE by flow cytometry. The ability to block small substrate cleavage by HNE ranged from 0% (265-3K1) to 15-18% (39A and 203) to 100% (NP57). All antibodies had the ability to capture free HNE with varying degrees of sensitivity, but HNE neutralisation by AAT resulted in complete loss of detection (NP57) to 2-4-fold decreased detection (39A and 203) to a 8-fold increase in detection (265-3K1). None of the monoclonal antibodies could detect 200 ng of free HNE, or HNE in complex with AAT, by Western blot analysis, which was easily detected by polyclonal antibodies. NP57 and 265-3K1 gave 10-fold higher fluorescence when detecting intracellular HNE than 39A and 203, and intracellular fluorescence decreased by 10-28% following maximal stimulation of purified neutrophils with fMLP and cytochalasin B (compared to 40% release determined by functional assay). However, for sub-maximal stimulation of neutrophils intracellular anti-HNE antibody binding increased, likely due to increased accessibility following redistribution of enzyme, indicating that measuring residual intracellular HNE as an index of release is a less reliable method than directly measuring extracellular HNE.

The interrupter technique to assess airway responsiveness in children with cystic fibrosis.

The aim of this study was to assess the validity of the interrupter technique (Rint) in measuring airway responsiveness in children with cystic fibrosis. Fifty children (aged 6-16 years) with cystic fibrosis performed six Rint measurements followed by three acceptable forced expiratory maneuvers. Each child then inhaled 5 mg of nebulized salbutamol by facemask. After 20 min the Rint and forced expiratory measurements were repeated. In the population as a whole a moderate but significant correlation between inverse Rint and FEV1 values was observed, both before and after inhaled bronchodilator (r=0.71 and 0.72, respectively, P < 0.001). However, when changes in Rint and FEV1 readings following inhaled bronchodilator were examined, no relationship was seen. Indeed, the two methods identified completely different subsets of children as being bronchodilator responsive. These results indicate that although a relationship exists between Rint and FEV1 in the whole population, this is not the case in individual children. Rint and FEV1 reflect different aspects of lung function. It is not appropriate to use Rint as a simple alternative for FEV1 in children with cystic fibrosis when assessing airway responsiveness.

The role of inflammation and infection in the development of chronic lung disease of prematurity.

CLD is a significant cause of infant morbidity and mortality. The lung injury is multifactorial in origin with supplemental oxygen and ventilatory damage being only part of the picture. Antenatal and postnatal infection and inflammation are also important in the development of CLD, although their precise role has still to be fully ascertained. In the future, therapeutic strategies need to be considered to decrease the incidence and severity of CLD. In particular a definitive trial investigating the role of antibiotics against Ureaplasma urealyticum in preventing CLD needs to be performed. Increased use of newer microbiological methods will also improve our understanding of the role of infection in CLD and further guide research and clinical management.

Antenatal infection and inflammation: what's new?

PURPOSE OF REVIEW: Recently antenatal infection and inflammation have received significant attention because of their potential role in promoting premature labour and in causing morbidity in preterm infants including the development of chronic lung disease of prematurity and cerebral white matter changes. This review highlights recent developments in this field. RECENT FINDINGS: Recent reports have improved our understanding of the inflammatory responses to antenatal infection although questions remain about the exact relationship between antenatal infection and inflammation. Although microbial invasion of the intrauterine cavity is confirmed, strategies to prevent onset of premature labour have not been successful. The association between antenatal infection and development of chronic lung disease has been confirmed and many mechanisms further explored for development of chronic lung disease and cerebral white matter changes. Ureaplasma species have been revisited for their role in both preterm labour and neonatal morbidity, especially as modern molecular methods have eased their identification. SUMMARY: Antenatal infection and inflammation are strongly implicated in the pathogenesis of preterm labour and in the development of neonatal morbidity, specifically chronic lung disease and white matter damage. Ureaplasma species are being increasingly studied for their role in these disorders. The challenge is to develop effective therapies to prevent these conditions.