Long-Term Pulmonal Therapy of Cystic Fibrosis-Patients with Amitriptyline

Background/Aims: Several recent clinical studies revealed an accumulation of ceramide in bronchial epithelial cells of patients with cystic fibrosis (CF). Degradation of ceramide concentrations in lungs of CF patients employing the functional acid sphingomyelinase inhibitor amitriptyline revealed a benefit in lung function, weight and exacerbation rates. Methods: To test for a beneficial effect of amitriptyline in vivo, we performed two phase II randomised, double-blind, placebo-controlled studies. CF patients were treated with 25 mg amitriptyline twice daily, i.e. a total dose of 50 mg/d. After those two studies part of the patients used amitriptyline in an off-lable-use for routine treatment. These patients were observed after one, two and three years after continuous use of amitriptyline and were matched with those patients who were not treated. These patients were used as a control group. Results: After one year of treatment, forced expiratory volume in 1 sec predicted (FEV1) increased significantly by 7.6±7.0%, p=1 decreased significantly in the control group by 1.8±3.3%, p=0.010, and weight increased by 1.1±2.7kg, p=0.010 (n=14). After two years of treatment, FEV1 increased significantly by 5.6±10.3%, p=0.009, and weight increased by 3.6±2.9kg, p=1 decreased in the control group by 2.1±3.7%, p=0.051 and weight increased by only 0.4±2.9kg, p=0.31 (n=10). After three years of treatment, FEV1 increased significantly by 7.7±8%, p=0.050, and weight increased by 7.3±3.8kg, p=0.016, in the amitriptyline population (n=5), whereas FEV1 decreased in the control group by 1.0±1.3%, p=0.075 and weight increased by 0.4±1.5kg, p=0.29 (n=5). Conclusion: Amitriptyline significantly increases FEV1, reduces ceramide in lung cells and increases weight of CF patients

Proteolytic Origin of the Soluble Human IL-6R In Vivo and a Decisive Role of N-Glycosylation.

Signaling of the cytokine interleukin-6 (IL-6) via its soluble IL-6 receptor (sIL-6R) is responsible for the proinflammatory properties of IL-6 and constitutes an attractive therapeutic target, but how the sIL-6R is generated in vivo remains largely unclear. Here, we use liquid chromatography-mass spectrometry to identify an sIL-6R form in human serum that originates from proteolytic cleavage, map its cleavage site between Pro-355 and Val-356, and determine the occupancy of all O- and N-glycosylation sites of the human sIL-6R. The metalloprotease a disintegrin and metalloproteinase 17 (ADAM17) uses this cleavage site in vitro, and mutation of Val-356 is sufficient to completely abrogate IL-6R proteolysis. N- and O-glycosylation were dispensable for signaling of the IL-6R, but proteolysis was orchestrated by an N- and O-glycosylated sequon near the cleavage site and an N-glycan exosite in domain D1. Proteolysis of an IL-6R completely devoid of glycans is significantly impaired. Thus, glycosylation is an important regulator for sIL-6R generation

ADAM17 and ADAM10 cleave the IL-6R between Pro-355 and Val-356 in vitro.

<p>(A) A schematic illustration, a representative coomassie-stained SDS gel, and a western blot of the precipitated protein after PMA-induced ADAM17-mediated sIL-6R generation are shown. The SDS gels were run under reducing conditions (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000080#sec013" target="_blank">Materials and Methods</a> for details), and the region of the SDS gel excised for MS is indicated with a red box. The precipitated sIL-6R is detected with an antibody that specifically recognizes the ectodomain of the human IL-6R (4–11). (B) MS/MS spectra (HCD) of the C-terminal peptide of the sIL-6R identified by manual spectra interpretation. One of the identified O-glycan structures on Thr-352 is shown. (C) A schematic illustration, a representative coomassie-stained SDS gel, and a western blot of the precipitated protein after ionomycin-induced ADAM10-mediated sIL-6R generation are shown. The SDS gels and the western blot were performed as described under panel (A). (D) MS/MS spectra (HCD) of the C-terminal peptide of the sIL-6R identified by manual spectra interpretation. One of the identified O-glycan structures on Thr-352 is shown.</p

An IL-6R devoid of all N-glycans is defective in ADAM17-mediated proteolysis.

<p>(A) PMA-mediated ectodomain shedding of IL-6R-5N with a stably transduced Ba/F3-gp130 cell line. Data shown are the mean ± SD from three independent experiments compared to wild-type IL-6R. (B) HEK293 cells were transiently transfected with a cDNA encoding wild-type IL-6R. Cells were pretreated with the ADAM10-specific inhibitor GI or the combined ADAM10/ADAM17-specific inhibitor GW for 30 min and then stimulated with 100 nM PMA for 2 h as indicated. The amount of sIL-6R in the cell supernatant was determined by ELISA. One out of three experiments with similar outcomes is shown (mean ± SD, <i>n</i> = 3). (C) The experiment was performed as described under (B), but sIL-6R was precipitated from cell supernatant and analyzed by western blot. Furthermore, the cells were lysed, and IL-6R expression in the lysates was also determined by western blot. β-actin served as loading control. One experiment out of three with similar outcomes is shown. (D, E) The experiment was performed as described in (C) and (D), but HEK293 cells were transiently transfected with a cDNA encoding IL-6R-5N. nd, not detected. (F) Cell-surface expression of the IL-6R-5N variant on transiently transfected HEK293 cells analyzed via flow cytometry (black histogram). The control staining is shown in gray. (G) HEK293 cells were transiently transfected with expression plasmids encoding wild-type IL-6R or IL-6R-5N in combination with either an expression plasmid encoding mCherry or MPD17-CANDIS. After 48 h, cells were lysed and the MPD17-CANDIS precipitated via its protein C (PC) tag. The expression of all proteins (input) and the interaction of the IL-6R variants with MPD17-CANDIS were analyzed via western blot. One experiment out of two with similar outcomes is shown.</p

N- and O-linked glycosylation are dispensable for intracellular transport and signaling of the IL-6R.

<p>(A–G) Equal numbers of Ba/F3-gp130 cells were incubated for 48 h with increasing amounts (0–100 ng/ml) of either IL-6 or Hyper-IL-6. The stably transduced IL-6R variant is indicated above the diagram. One representative experiment out of three performed is shown (mean ± SD, biological triplicates). (H) Equal numbers of Ba/F3-gp130-hIL-6R and Ba/F3-gp130-hIL-6R-4N cells were labeled with an anti-IL-6R antibody and incubated at 37°C for the indicated time periods. The remaining cell-surface expression of the IL-6R was analyzed via flow cytometry. Data shown are the mean ± SD from three independent experiments.</p

Mutation of Val-356 is sufficient to block proteolysis of the IL-6R and the Asp358Ala variant.

<p>(A) Amino-acid residues from Asp-340 to Ala-370 of the human IL-6R. The identified cleavage site between Pro-355 (P1) and Val-356 (P1′) is indicated. (B) Overview of the six different IL-6R cleavage site mutants. Mutations are colored in either green or blue; the amino-acid residues of the wild-type cleavage site are shown in red. (C) HEK293 cells were transiently transfected with expression plasmids encoding the wild-type IL-6R (PV) or the double mutants (IE, DG) as indicated. Cells were either treated with 100 nM PMA for 2 h or DMSO as vehicle control. sIL-6R was precipitated from the supernatant with concanavalin A-covered sepharose beads, and the cells were lysed. Both were analyzed via western blot, and β-actin served as the loading control. One out of three experiments with similar outcomes is shown. (D, E) The experiment was performed as described under (C), but sIL-6R generation was analyzed via ELISA. In (D), the amount of sIL-6R generated after PMA stimulation of the wild-type IL-6R (PV) was set to 100%, and all other values were calculated accordingly. In (E), the amount of sIL-6R without stimulation was considered as constitutive shedding and set to 1, and the increase of sIL-6R was calculated. Data shown are the mean ± SD from at least three independent experiments (*<i>p</i> < 0.05, ns = no significant difference). (F–H) HEK293 cells were transiently transfected with expression plasmids encoding the wild-type IL-6R (PV) or the single mutants (DV, IV, PE, PG) as indicated. The experiments were performed as described in (C) to (E). (I) Overview of the four different IL-6R cleavage site mutants of the IL-6R Asp358Ala variant. Mutations are colored in either green or blue, the amino-acid residues of the wild-type cleavage site are shown in red, and the Asp358Ala single nucleotide polymorphism (SNP) is colored in orange. (J) ADAM17-mediated proteolysis of the IL-6R variants depicted in (I) were analyzed as described in (D). (K–M) Equal numbers of Ba/F3-gp130 cells were incubated for 48 h with increasing amounts (0–100 ng/ml) of either IL-6 or Hyper-IL-6. The stably transduced IL-6R variant is indicated above the diagram. One representative experiment out of three performed is shown (mean ± SD, biological triplicates).</p