High concentrations of pepsin in bronchoalveolar lavage fluid from children with cystic fibrosis are associated with high interleukin-8 concentrations.

BACKGROUND: Gastro-oesophageal reflux is common in children with cystic fibrosis (CF) and is thought to be associated with pulmonary aspiration of gastric contents. The measurement of pepsin in bronchoalveolar lavage (BAL) fluid has recently been suggested to be a reliable indicator of aspiration. The prevalence of pulmonary aspiration in a group of children with CF was assessed and its association with lung inflammation investigated. METHODS: This was a cross-sectional case-control study. BAL fluid was collected from individuals with CF (n=31) and healthy controls (n=7). Interleukin-8 (IL-8), pepsin, neutrophil numbers and neutrophil elastase activity levels were measured in all samples. Clinical, microbiological and lung function data were collected from medical notes. RESULTS: The pepsin concentration in BAL fluid was higher in the CF group than in controls (mean (SD) 24.4 (27.4) ng/ml vs 4.3 (4.0) ng/ml, p=0.03). Those with CF who had raised pepsin concentrations had higher levels of IL-8 in the BAL fluid than those with a concentration comparable to controls (3.7 (2.7) ng/ml vs 1.4 (0.9) ng/ml, p=0.004). Within the CF group there was a moderate positive correlation between pepsin concentration and IL-8 in BAL fluid (r=0.48, p=0.04). There was no association between BAL fluid pepsin concentrations and age, sex, body mass index z score, forced expiratory volume in 1 s or Pseudomonas aeruginosa colonisation status. CONCLUSIONS: Many children with CF have increased levels of pepsin in the BAL fluid compared with normal controls. Increased pepsin levels were associated with higher IL-8 concentrations in BAL fluid. These data suggest that aspiration of gastric contents occurs in a subset of patients with CF and is associated with more pronounced lung inflammation.

Anti-apoptotic effects of Z alpha1-antitrypsin in human bronchial epithelial cells.

alpha(1)-antitrypsin (alpha(1)-AT) deficiency is a genetic disease which manifests as early-onset emphysema or liver disease. Although the majority of alpha(1)-AT is produced by the liver, it is also produced by bronchial epithelial cells, amongst others, in the lung. Herein, we investigate the effects of mutant Z alpha(1)-AT (ZAAT) expression on apoptosis in a human bronchial epithelial cell line (16HBE14o-) and delineate the mechanisms involved. Control, M variant alpha(1)-AT (MAAT)- or ZAAT-expressing cells were assessed for apoptosis, caspase-3 activity, cell viability, phosphorylation of Bad, nuclear factor (NF)-kappaB activation and induced expression of a selection of pro- and anti-apoptotic genes. Expression of ZAAT in 16HBE14o- cells, like MAAT, inhibited basal and agonist-induced apoptosis. ZAAT expression also inhibited caspase-3 activity by 57% compared with control cells (p = 0.05) and was a more potent inhibitor than MAAT. Whilst ZAAT had no effect on the activity of Bad, its expression activated NF-kappaB-dependent gene expression above control or MAAT-expressing cells. In 16HBE14o- cells but not HEK293 cells, ZAAT upregulated expression of cIAP-1, an upstream regulator of NF-kappaB. cIAP1 expression was increased in ZAAT versus MAAT bronchial biopsies. The data suggest a novel mechanism by which ZAAT may promote human bronchial epithelial cell survival.

Alpha-1-antitrypsin aerosolised augmentation abrogates neutrophil elastase-induced expression of cathepsin B and matrix metalloprotease 2 in vivo and in vitro.

BACKGROUND: Neutrophil elastase (NE) activity is increased in lung diseases such as alpha(1)-antitrypsin (A1AT) deficiency and pneumonia. It has recently been shown to induce expression of cathepsin B and matrix metalloprotease 2 (MMP-2) in vitro and in a mouse model. It is postulated that increased cathepsin B and MMP-2 in acute and chronic lung diseases result from high levels of extracellular NE and that expression of these proteases could be inhibited by A1AT augmentation therapy. METHODS: Cathepsin and MMP activities were assessed in bronchoalveolar lavage (BAL) fluid from patients with A1AT deficiency, pneumonia and control subjects. Macrophages were exposed to BAL fluid rich in free NE from patients with pneumonia following pretreatment with A1AT. MMP-2, cathepsin B, secretory leucoprotease inhibitor (SLPI) and lactoferrin levels were determined in BAL fluid from A1AT-deficient patients before and after aerosolisation of A1AT. RESULTS: BAL fluid from both patients with pneumonia and those with A1AT deficiency containing free NE had increased cathepsin B and MMP-2 activities compared with BAL fluid from healthy volunteers. The addition of A1AT to BAL fluid from patients with pneumonia greatly reduced NE-induced cathepsin B and MMP-2 expression in macrophages in vitro. A1AT augmentation therapy to A1AT-deficient individuals also reduced cathepsin B and MMP-2 activity in BAL fluid in vivo. Furthermore, A1AT-deficient patients had higher levels of SLPI and lactoferrin after A1AT augmentation therapy. CONCLUSION: These findings suggest a novel role for A1AT inhibition of NE-induced upregulation of MMP and cathepsin expression both in vitro and in vivo.

Alpha-1 antitrypsin deficiency: a conformational disease associated with lung and liver manifestations.

Alpha-1 antitrypsin (A1AT) is a serine anti-protease produced chiefly by the liver. A1AT deficiency is a genetic disorder characterized by serum levels of less than 11 mumol/L and is associated with liver and lung manifestations. The liver disease, which occurs in up to 15% of A1AT-deficient individuals, is a result of toxic gain-of-function mutations in the A1AT gene, which cause the A1AT protein to fold aberrantly and accumulate in the endoplasmic reticulum of hepatocytes. The lung disease is associated with loss-of-function, specifically decreased anti-protease protection on the airway epithelial surface. The so-called 'Z' mutation in A1AT deficiency encodes a glutamic acid-to-lysine substitution at position 342 in A1AT and is the most common A1AT allele associated with disease. Here we review the current understanding of the molecular pathogenesis of A1AT deficiency and the best clinical management protocols.

A secretory leukocyte protease inhibitor variant with improved activity against lung infection.

Secretory leukocyte protease inhibitor (SLPI) is an important respiratory tract host defense protein, which is proteolytically inactivated by excessive neutrophil elastase (NE) during chronic Pseudomonas infection in the cystic fibrosis (CF) lung. We generated two putative NE-resistant variants of SLPI by site-directed mutagenesis, SLPI-A16G and SLPI-S15G-A16G, with a view to improving SLPI's proteolytic stability. Both variants showed enhanced resistance to degradation in the presence of excess NE as well as CF patient sputum compared with SLPI-wild type (SLPI-WT). The ability of both variants to bind bacterial lipopolysaccharides and interact with nuclear factor-κB DNA binding sites was also preserved. Finally, we demonstrate increased anti-inflammatory activity of the SLPI-A16G protein compared with SLPI-WT in a murine model of pulmonary Pseudomonas infection. This study demonstrates the increased stability of these SLPI variants compared with SLPI-WT and their therapeutic potential as a putative anti-inflammatory treatment for CF lung disease.

Type-I interferons induce lung protease responses following respiratory syncytial virus infection via RIG-I-like receptors.

The role of proteases in viral infection of the lung is poorly understood. Thus, we examined matrix metalloproteinases (MMPs) and cathepsin proteases in respiratory syncytial virus (RSV)-infected mouse lungs. RSV-induced gene expression for MMPs -2, -3, -7, -8, -9, -10, -12, -13, -14, -16, -17, -19, -20, -25, -27, and -28 and cathepsins B, C, E, G, H, K, L1, S, W, and Z in the airways of Friend leukemia virus B sensitive strain mice. Increased proteases were present in the bronchoalveolar lavage fluid (BALF) and lung tissue during infection. Mitochondrial antiviral-signaling protein (MAVS) and TIR-domain-containing adapter-inducing interferon-ß-deficient mice were exposed to RSV. Mavs-deficient mice had significantly lower expression of airway MMP-2, -3, -7, -8, -9, -10, -12, -13, and -28 and cathepsins C, G, K, S, W, and Z. In lung epithelial cells, retinoic acid-inducible gene-1 (RIG-I) was identified as the major RIG-I-like receptor required for RSV-induced protease expression via MAVS. Overexpression of RIG-I or treatment with interferon-ß in these cells induced MMP and cathepsin gene and protein expression. The significance of RIG-1 protease induction was demonstrated by the fact that inhibiting proteases with batimastat, E64 or ribavirin prevented airway hyperresponsiveness and enhanced viral clearance in RSV-infected mice.

The role of secretory leucoprotease inhibitor in the resolution of inflammatory responses.

Chronic lung disease is one of the most common causes of death and disability worldwide. This group of diseases is characterized by a protease burden, an infective process and a dominant pro-inflammatory profile. While SLPI (secretory leucoprotease inhibitor) was initially identified as a serine protease inhibitor, it has since been shown that SLPI possesses other properties distinct from those associated with its antiprotease capabilities that play an important role in protecting the host from infection and injury. In the course of this review, we will highlight the findings from a range of studies that illustrate the multiple functions of SLPI and its role in the resolution of the immune response.

Role of IL-18 in CD4+ T lymphocyte activation in sarcoidosis.

Sarcoidosis is a granulomatous disease of unknown etiology associated with the expansion of IL-2-producing activated CD4(+) T lymphocytes. A number of factors including the recently described IL-18 have been implicated in IL-2 expression in vitro. We investigated the role of IL-18 in IL-2 expression in sarcoidosis. Eighteen individuals with sarcoidosis and 15 normal controls were studied. IL-18R expression and epithelial lining fluid (ELF) concentrations of IL-18 were significantly elevated in the sarcoid group (p = 0.0143 and 0.0024, respectively). Both AP1 and NF-kappaB, transcription factors that regulate IL-2 gene expression, were activated in vivo in sarcoid pulmonary CD4(+) T lymphocytes. Transcription factor activity was not detected in pulmonary CD4(+) T lymphocytes from normal controls or from peripheral blood CD4(+) T lymphocytes from individuals with sarcoidosis, further evidence of compartmentalization of the lymphoproliferative process in this condition. We examined the effects of IL-18 on AP1 and NF-kappaB in Jurkat T cells in vitro. These effects were both time and dose dependent. Examination of transcription factor activation and IL-2 gene expression in Jurkat T cells revealed that sarcoid but not normal ELF activated AP1 and NF-kappaB, induced IL-2 gene transcription, and up-regulated IL-2 protein production. Addition of IL-18 to normal ELF also induced IL-2 mRNA accumulation, whereas correspondent depletion of IL-18 from sarcoid ELF using neutralizing Abs abrogated all of the effects. These data strongly implicate IL-18 in the pathogenesis of sarcoidosis via activation of AP1 and NF-kappaB, leading to enhanced IL-2 gene expression and IL-2 protein production and concomitant T cell activation.

Cathepsin B, L, and S cleave and inactivate secretory leucoprotease inhibitor.

A number of serine proteases, matrix metalloproteases, and cysteine proteases were evaluated for their ability to cleave and inactivate the antiprotease, secretory leucoprotease inhibitor (SLPI). None of the serine proteases or the matrix metalloproteases examined cleaved the SLPI protein. However, incubation with cathepsins B, L, and S resulted in the cleavage and inactivation of SLPI. All three cathepsins initially cleaved SLPI between residues Thr(67) and Tyr(68). The proteolytic cleavage of SLPI by all three cathepsins resulted in the loss of the active site of SLPI and the inactivation of SLPI anti-neutrophil elastase capacity. Cleavage and inactivation were catalytic with respect to the cathepsins, so that the majority of a 400-fold excess of SLPI was inactivated within 15 min by cathepsins L and S. Analysis of epithelial lining fluid samples from individuals with emphysema indicated the presence of cleaved SLPI in these samples whereas only intact SLPI was observed in control epithelial lining fluid samples. Active cathepsin L was shown to be present in emphysema epithelial lining fluid and inhibition of this protease prevented the cleavage of recombinant SLPI added to emphysema epithelial lining fluid. Taken together with previous data that demonstrates that cathepsin L inactivates alpha(1)-antitrypsin, these findings indicate the involvement of cathepsins in the diminution of the lung antiprotease screen possibly leading to lung destruction in emphysema.

Interleukin-8 up-regulation by neutrophil elastase is mediated by MyD88/IRAK/TRAF-6 in human bronchial epithelium.

Cystic fibrosis is characterized in the lungs by neutrophil-dominated inflammation mediated significantly by neutrophil elastase (NE). Previous work has shown that NE induces interleukin-8 (IL-8) gene expression and protein secretion in bronchial epithelial cells. We sought to determine the intracellular mechanisms by which NE up-regulates IL-8 in bronchial epithelial cells. The data show that stimulation of 16HBE14o(-) cells with NE induced IL-8 protein production and gene expression. Both responses were abrogated by actinomycin D, indicating that regulation is at the transcriptional level. Electrophoretic mobility shift assays demonstrated that nuclear factor kappaB (NFkappaB) was activated in 16HBE14o(-) cells stimulated with NE. Western blot analysis demonstrated that activation of NFkappaB by NE was preceded by phosphorylation and degradation of IkappaB proteins, principally IkappaBbeta. In addition, we observed that interleukin-1 receptor-associated kinase (IRAK) was degraded in 16HBE14o(-) cells stimulated with NE. Quantification of IL-8 reporter gene activity by luminometry demonstrated that dominant negative MyD88 (MyD88Delta) or TRAF-6 (TRAF-6Delta) inhibited IL-8 reporter gene expression in response to NE. Furthermore, MyD88Delta inhibited NE-induced IRAK degradation. These results show that NE induces IL-8 gene up-regulation in bronchial epithelial cells through an IRAK signaling pathway involving both MyD88 and TRAF-6, resulting in degradation of IkappaBbeta and nuclear translocation of NFkappaB. These findings may have implications for therapeutic treatments in the cystic fibrosis condition.