Effect of Chondroitinase ABC on Purulent Sputum from Cystic Fibrosis and Other Patients

Abstract

Cystic fibrosis (CF) patients develop chronic lung infections associated with airway obstruction by viscous and insoluble mucus secretions. Although mucus glycoproteins (mucins) are thought to be responsible for mucus plugs, other glycoconjugate components of airway secretions have not been systematically evaluated. The aim of the present study was to determine whether chondroitin sulfate proteoglycans (CSPG) contribute to the insolubility of CF sputum. Sputa obtained from 18 CF patients were incubated with chondroitinase ABC (ChABC) or buffer (control) for 18 h at 37°C, and after centrifugation at 12,000 g, the volume of the insoluble pellet and turbidity of the supernatant were determined as measures of solubility. ChABC caused a 70 -90% reduction in supernatant turbidity and a 60 -70% decrease in pellet volume of the 13 purulent CF sputa, but had much less effect on the five nonpurulent CF sputa tested. Similar results were obtained with two non-CF purulent and two non-CF, nonpurulent sputa. Gel electrophoresis, Western blot, and slot blot immunoassays with antichondroitin sulfate and antimucin antibodies revealed that purulent sputa (CF and non-CF) contained more CSPG and less mucin than nonpurulent sputa. In vitro mixing experiments showed that mucin in nonpurulent sputa was reduced upon incubation with purulent sputa, presumably because of degradation or a loss of immunoreactive mucin epitopes from leukocyte and/or bacterial enzymes present in purulent sputa. Our results suggest that CSPG contribute more significantly than mucins to the insolubility of purulent tracheobronchial secretions from CF patients. Because purulent sputa from non-CF patients showed a similar pattern, our observations with CF sputa may have wider applicability. CF is a life-threatening inherited disease caused by genetic mutation of the CF transmembrane conductance regulator (CFTR), a cyclic AMP-dependent chloride channel present in secretory and other cells throughout the body. Despite rapid advances in our knowledge of the structure and function of CFTR, the link between the mutations in this gene and the clinical manifestation of widespread obstruction of exocrine glands with viscous mucus secretions remains obscure.Almost all CF patients develop chronic lung disease related to airway obstruction, inflammation, and recurrent infections that are eventually lethal. In CF airways and intestine, goblet cell hyperplasia and mucin hypersecretion are characteristic, as is the presence of dilated airway submucous glands and intestinal crypt lumina (1-4). Inflammatory cell products stimulate airway secretions and thus contribute to mucus obstruction (4). Secretory mucin macromolecules, consisting of disulfidedependent polymers secreted by epithelial cells, are thought to be largely responsible for the viscoelastic gels and obstructive plugging of bronchial secretions. Another component that contributes to increased viscosity is DNA released from lysed neutrophils in the airways (5,6). This has led to the clinical use of recombinant DNase ...

IL-8 Dictates Glycosaminoglycan Binding and Stability of IL-18 in Cystic Fibrosis

“…For example, a reduced level of HS and increased CS has been observed in atherosclerosis (51). Increased sulphation and concentrations of GAGs (52,53) have been found in BALF from children with CF, and secretion of HS (13), CS (26,54), and HA (27,55) is markedly increased in bronchial cells and CF tissues. Increased expression of GAGs (including HS) at the endothelium in CF may point to an exceptional characteristic of CF that contributes to increased stability of IL-8, thus leading to chronic neutrophil infiltration and inflammation (13).…”

Disruption of Tight Junctions during Traversal of the Respiratory Epithelium by Burkholderia cenocepacia

“…Polarized epithelial cells incubated with medium alone or infected with B. cenocepacia for 24 h were washed, fixed in methanol at Ϫ20°C, and blocked with 1% BSA for 1 h. The fixed cells were thereafter incubated overnight at 4°C with a mouse monoclonal antibody to either ZO-1 or occludin (BD Biosciences). In addition, B. cenocepacia was colocalized with these tightjunction proteins by probing the cell layers with a previously described polyclonal rabbit antibody (10,16). After the cell layers were washed to remove unbound antibody, they were incubated with Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin G (Molecular Probes Inc., Eugene, OR) to detect ZO-1 and occludin or Alexa Fluor 594-conjugated goat anti-rabbit immunoglobulin G (Molecular Probes) to visualize B. cenocepacia.…”

Responses of Well-Differentiated Airway Epithelial Cell Cultures from Healthy Donors and Patients with Cystic Fibrosis to Burkholderia cenocepacia Infection

“…A monoclonal antibody to ␤-tubulin conjugated with the fluorophore indocarbocyanine was obtained from Sigma Chemical Co. (Burlington, Canada). Polyclonal antibodies to B. cenocepacia (designated R418) and tracheobronchial mucin have been described previously (14,33).…”