S.L. Martin

Association of airway cathepsin B and S with inflammation in cystic fibrosis.

Irreversible tissue damage within the cystic fibrosis (CF) lung is mediated by proteolytic enzymes during an inflammatory response. Serine proteinases, in particular neutrophil elastase (NE), have been implicated however, members of the cysteine proteinase family may also be involved. The aim of this study was to determine cathepsin B and S levels in cystic fibrosis (CF) sputum and to assess any relationship to recognized markers of inflammation such as sputum NE, interleukin‐8 (IL‐8), tumor necrosis factor alpha (TNF‐α), urine TNF receptor 1 (TNFr1), plasma IL‐6, and serum C‐reactive protein (CRP). Proteinase activities were measured in the sputum of 36 clinically stable CF patients using spectrophotometric and fluorogenic assays. Immunoblots were also used to confirm enzyme activity data. All other parameters were measured by ELISA. Patients had a mean age of 27.2 (8.2) years, FEV. of 1.6 (0.79) L and BMI of 20.7 (2.8). Both cathepsin B and S activities were detected in all samples, with mean concentrations of 18.0 (13.5) µg/ml and 1.6 (0.88) µg/ml, respectively and were found to correlate not only with each other but with NE, TNF‐α and IL‐8 (in all cases . < 0.05). Airway cathepsin B further correlated with circulatory IL‐6 and CRP however, no relationship for either cathepsin was observed with urine TNFr1. This data indicates that cathepsin B and S may have important roles in the pathophysiology of CF lung disease and could have potential as markers of inflammation in future studies. Pediatr. Pulmonol. 2010; 45:860–868.

Cytokine concentrations and neutrophil elastase activity in bronchoalveolar lavage and induced sputum from patients with cystic fibrosis, mild asthma and healthy volunteers

BACKGROUND Induced sputum (IS) has been proposed as a non-invasive alternative to bronchoalveolar lavage (BAL) for the assessment and monitoring of airways inflammation. The aim of this study was to compare both methods in patients with cystic fibrosis (CF). The possible differences between subjects with CF, mild asthma and healthy volunteers (HV) was also assessed. METHOD In a single centre, randomised, two way crossover study, 11 patients with CF, 9 mild asthmatics (MA) and 11 HV underwent BAL and hypertonic saline induction on consecutive days. Free neutrophil elastase (NE), neutrophil elastase/alpha(1)-anti-trypsin complex (NE-AAT), tumour necrosis factor receptor (p55) and interleukin-8 (IL-8) were measured in cell free supernatants. RESULTS Three CF patients reported serious adverse events following BAL. NE was usually undetectable in both IS or BAL samples and NE-AAT concentrations did not differ consistently between the two sampling methods. IL-8 and p55 levels in the CF patients tended to be higher in IS samples compared with BAL samples (median 19,860 vs. 3,855 pg/ml and 2.55 vs. 0.29 ng/ml, respectively). There was a significant difference in mean p55 concentrations between CF, MA and HV in IS samples (P=0.003) but not in BAL samples (P=0.36). The difference in mean IL-8 concentrations in IS samples between subject groups was statistically different (P=0.023). CONCLUSIONS IS samples can be safely obtained from CF patients. Analysis of IS samples can help to characterize the inflammatory process in the airways of CF patients. The serious adverse events following BAL in 3 CF patients highlight an inherent risk associated with this procedure.

Inflammatory and immunological biomarkers are not related to survival in adults with Cystic Fibrosis

BACKGROUND Chronic Pseudomonas aeruginosa pulmonary infection is associated with a decline in lung function and reduced survival in people with Cystic Fibrosis (CF). Damaging inflammatory and immunological mediators released in the lungs can be used as markers of chronic infection, inflammation and lung tissue damage. METHODS Clinical samples were collected from CF patients and healthy controls. Serum IgG and IgA anti-Pseudomonas antibodies, sputum IL-8 and TNFα, plasma IL-6 and urine TNFr1 were measured by ELISA. Sputum neutrophil elastase (NE), cathepsin S and cathepsin B were measured by spectrophotometric and fluorogenic assays. The relationship between IgG and IgA, inflammatory mediators and long-term survival was determined. RESULTS IgG and IL-6 positively correlated with mortality. However, multivariate analysis demonstrated that after adjusting for FEV(1), IgG was not independently related to mortality. A relationship was observed between IgG and IL-6, TNFα, TNFr1 and between IgA and IL8, cathepsin S and cathepsin B. CONCLUSIONS These data indicate that biomarkers of inflammation are not independent predictors of survival in people with CF.

Chymotrypsin-like serine proteinases are involved in the maintenance of cell viability

An increasing number of studies have implicated serine proteinases in the development of apoptosis. In this study, we assessed the ability of a set of highly specific irreversible inhibitors (activity probes), incorporating an α-amino alkane diphenyl phosphonate moiety, to modulate cell death. In an initial assessment of the cellular toxicity of these activity probes, we discovered that one example, N-α-tetramethylrhodamine phenylalanine diphenylphosphonate {TMR-Phe(P)(OPh)(2)} caused a concentration-dependent decrease in the viability of HeLa and U251 mg cells. This reduced cell viability was associated with a time-dependent increase in caspase-3 activity, PARP cleavage and phosphatidylserine translocation, establishing apoptosis as the mechanism of cell death. SDS-PAGE analysis of cell lysates prepared from the HeLa cells treated with TMR-Phe(P)(OPh)(2), revealed the presence of a fluorescent band of molecular weight 58 kDa. Given that we have previously reported on the use of this type of activity probe to reveal active proteolytic species, we believe that we have identified a chymotrypsin-like serine proteinase activity integral to the maintenance of cell viability.

Degradation of host defence molecules by CF-related pathogens grown as biofilms

We investigated the ability of secreted bacterial proteinases from three pathogens [Burkholderia multivorans (Bm), Burkholderia cenocepacia (Bc), and Pseudomonas aeruginosa (Pa)] involved in chronic bacterial infections in cystic fibrosis to degrade various host defence-related molecules. These included two endogenous proteinase inhibitors, secretory leukocyte proteinase inhibitor (rhSLPI) and alpha-1 antitrypsin (AAT); two relevant immunoglobulins, secretory IgA (sIgA) and IgG, and two proteins important in innate immunity, lactoferrin and lysozyme. Host defence-related molecules were co-incubated with cell-free supernatants from 48 hour biofilm cultures, grown on mucin-coated microplates, from all three pathogens under investigation (six isolates from each species). No degradation of AAT, sIgA, IgG, and lactoferrin was observed for any of the organisms. Of the 18 isolates tested, only one demonstrated the ability to degrade lysozyme. This was an environmental isolate of Pa which was included as a comparison for the predominantly clinically relevant isolates used in the study. In contrast, all isolates of Bm (n = 4) and Pa (n = 4) were able to degrade rhSLPI however, out of five bacterial isolates tested for Bc only two demonstrated a limited ability to degrade the molecule with >95% of the protein band still remaining intact at the end of the experiment. This study demonstrates that the majority of the host defence molecules investigated are resistant to degradation by bacterial proteinases from Bm, Bc and Pa when grown as a biofilm. However, rhSLPI was vulnerable to significant degradation which could result in aberrant serine proteolysis in regions of the lungs containing biofilm growth.

84 Characterisation of fibrinogen degradation by the CF-related pathogens, Burkholderia cenocepacia and Pseudomonas aeruginosa

Bacterial proteinases have been implicated in causing necrotic or hemorrhagic tissue damage. We investigated the ability of secreted bacterial proteinases, from two pathogens involved in chronic bacterial infections in cystic fibrosis, to degrade fibrinogen. We found that multiple proteinase species secreted by Burkholderia cenocepacia and Pseudomonas aeruginosa were able to degrade fibrinogen, and the degradation profile was different from that observed when fibrinogen was incubated with thrombin. Furthermore, we investigated the effect incorporation of broadspectrum and specific proteinase inhibitors would have on the ability of bacterial proteinases to degrade fibrinogen. Treatment of B. cenocepacia-fibrinogen cocultures with the individual inhibitors resulted in a partial inhibition of degradation. However, P. aeruginosa-fibrinogen co-cultures showed no differences in degradation profile when inhibitors were used singly. For both cultures total degradation inhibition was observed when the samples were treated with different inhibitor combinations. These results indicate that multiple bacterial proteinase species may play a role in the processes leading to the degradation of fibrinogen, which could impair polymerisation of fibrin, thus contributing to the excessive hemorrhagic tissue damage seen in the late stages of cystic fibrosis lung disease.