Figure 1.
The sputum of cystic fibrosis patients contains neutrophils undergoing NET formation.
(a) Representative fluorescence microscopy images of neutrophils undergoing NET formation in the sputum of CF patients. Samples were labeled with antibodies against NE (red) and MPO (green), and with the DNA dye Hoechst (blue). CF sputum contains: (i) intact naïve neutrophils. (ii, iii) neutrophils with decondensed chromatin where NE, MPO and DNA colocalize (arrows), (iii) large aggregates that contain DNA, MPO and NE (asterisk). Scale bar: 10 µm. (b, c) The distribution of intact neutrophils and neutrophils that have made NETs in 2 untreated patients (P1 and P2) and 2 patients receiving DNase therapy (P3 and P4). The estimates are based on a combination of microscopic and biochemical measurements. (b) Intact cells/mL (grey) measured by microscopy, and total cells – intact cells/mL (black). The total number of cells was estimated from MPO activity/mL of sputum measured against a lysate derived from a known number of neutrophils. A value of 104 µg MPO/106 neutrophils was used to estimate the total number of neutrophils (c) The data from (b) plotted as normalized distribution of cells.
Figure 2.
CF sputum contains DNA in complex with MPO, NE and histones.
(a) MPO and NE in CF sputum are predominantly bound to chromatin. Purified MPO, purified NE and solubilized sputum supernatant from a representative patient (3–8) digested with nucleases for 6 hrs in the absence of protease inhibitors, resolved by native agarose electrophoresis and blotted to nitrocellulose. MPO was detected by activity prior to gel transfer and by immunoblotting. NE, H3 and H4 were detected by immunoblotting. DNA was detected by ethidium bromide staining. Cartoon representation of the complexes (right) where DNA is depicted in blue and the different proteins are indicated. The cartoons are positioned next to the area of the gel where the corresponding proteins migrate. Similar results were obtained with samples from all CF patients. (b) NET content of CF sputum using the activity of MPO in comparison to the MPO activity of human NETs. The MPO levels in the soluble fraction of CF sputum solubilized with nucleases from 2 patients treated with Dornase were compared to MPO levels in MNase treated NETs from a known number of human neutrophils. Accordingly, 106 NETs treated with MNase gave rise to 0.92 µg soluble MPO. This ratio was used to obtain the NET equivalent/mL in CF sputum samples. (c) Comparison of the MPO levels in CF sputum by detection of MPO in supernatants solubilized with nucleases for 2 hrs, to the MPO content of 4.1×104 NETs, analyzed by native gel electrophoresis and western immunoblotting. (d) Quantitation of MPO levels in (c) using band digitization. (e) NET content in CF sputum based on native gel electrophoresis analysis of sputum supernatants solubilized with nucleases, by comparison of the band intensities in (c). All concentrations refer to the original sputum volume.
Figure 3.
NE enhances nuclease dependent sputum solubilization.
Time course analysis of untreated (black circles) and sputum treated with SLPI (open circles), or NEi (grey circles). (a) The sputum was solubilized by DNase administered to patients and MNase. Samples were incubated untreated or in the presence of NEi or SLPI. At the indicated timepoints, the samples were centrifuged at 1000× g for 10 min and the soluble volume was measured. The data are plotted as the % of soluble volume against the total volume of the sample and represent the average from individual patient measurements. (b) DNA is degraded in soluble sputum supernatants incubated with nucleases in the absence (black circles) or in the presence of SLPI (open circles) but not when incubated with NEi (grey circles). Soluble aliquots were obtained at the indicated timepoints and DNA was measured with the Quanti-T dsDNA assay. (c) NE activity in solubilized sputum supernatants over time. NE but not SLPI inhibits NE activity in CF sputum. (a, b) Data from a representative CF patient sample. The same trend was obtained in all patients but the overall levels of DNA and NE activity vary significantly amongst samples from different patients.
Figure 4.
NE degrades histones in CF sputum.
(a) The solubilized sputum samples were treated with Proteinase K to digest all proteins and were resolved by agarose gel electrophoresis. DNA was detected by ethidium bromide staining. The released DNA is approximately 200 bp long and is digested over time in samples where NE is active. NEi blocks the digestion of DNA after 24 hrs of incubation (asterisks). (b) MPO, NE and histones, solubilized from nuclease-treated CF sputum in vitro, are degraded over time. Protein degradation depends on the activity of NE. Immunoblot against MPO, histones H3 and H4 in sputum sample supernatants from one representative patient. Sputum samples from each patient in the absence or presence of NEi, or SLPI were resolved by SDS-PAGE electrophoresis. Aliquots were collected at the indicated time points during incubation with nucleases at 37°C. An NE/serpin complex appears (asterisk at approx. 60 kDa) in untreated and SLPI-treated sputum, which is distinct from NE alone (arrowhead). This band is approximately 55 kDa and co-migrates with α-1-anti-trypsin (A1AT) (asterisk). NEi blocks the formation of this higher molecular weight covalent complex between NE and A1AT, indicating that serpins are targeted and deactivated by active NE in CF sputum.
Figure 5.
NE, MPO and DNA form a complex in CF sputum.
(a) NE-mediated degradation of DNA-bound proteins enhances the degradation of DNA by exogenous nucleases and alters the migration pattern of MPO. Purified MPO or supernatants from sputum samples solubilized with nucleases at the indicated time points, either untreated (−) or treated with NEi (+NEi), were separated by electrophoresis on agarose gels under native conditions. A black circle with a (−) sign denotes the anode, and a red circle with the (+) denotes the cathode during electrophoresis. A line marks where the sample was loaded. MPO was detected by incubating the gel in a solution containing hydrogen peroxide and the MPO substrate O-phenylenediamine, which turns orange upon oxidation by the enzyme (middle panel). DNA was detected by ethidium bromide staining (right panel). The dependence of MPO migration on the presence of DNA and NE activity suggests that these molecules are part of the same macromolecular complex. No soluble sample could be collected at 2 hrs in the presence of NEi. (b) Low levels of DNA can be detected in excised gel slices containing MPO in (a) using the Quant-iT dsDNA assay. (a, b) Data from a representative CF patient sample.
Figure 6.
NE synergizes with therapeutic nucleases in promoting CF sputum solubilization.
By degrading histones, NE promotes the relaxation of chromatin which exposes DNA to the action of exogenously administered nucleases.