LL-37 levels and its ability to bind LPS in CF sputum are increased following treatment with DNase and heparinase II.

<p>(<b>A</b>) CF sputum samples (n = 12) were treated with Pulmozyme® DNase and/or heparinase II for 1 h at 37°C. The LL-37 content of these samples and untreated sputum samples were quantified by ELISA. (<b>B</b>) The ability of available LL-37 present in untreated and treated CF sputum samples to bind LPS was determined by ELISA. Results are expressed as percentage of untreated control (100% = untreated CF sputum). ** <i>p</i><0.01; *** <i>p</i><0.001.</p

LL-37 inhibits LPS-induced IL-8 production and IκB degradation in THP-1 monocytic cells.

<p>(<b>A</b>) THP-1 monocytes were pre-treated for 1 h with 10 µg/ml LL-37 and incubated in the absence or presence of 1 µg/ml <i>P. aeruginosa</i> LPS. After 24 h stimulation with LPS, IL-8 levels were quantified in cell-free culture supernatants by ELISA. *** <i>p</i><0.001; Con, control. (<b>B</b>) Cytoplasmic extracts were prepared following 30, 60 and 120 min and levels of IκBα, IκBβ, phosphorylation of IκBα (Ser32/36) and IKKα/β (Ser180/181) were determined by Western blotting. GAPDH was used to control for protein loading.</p

Increased LL-37 in DNase and heparinase II treated CF sputum inhibits IL-8 production from THP-1 monocytes.

<p>CF sputum samples (n = 12) were treated with Pulmozyme® DNase and/or heparinase II for 1 h at 37°C in the absence or presence of an LL-37 antibody or heat-inactivated (HI) LL-37 antibody as a negative control. Samples were incubated in LPS-immobilised 96-well microtiter plates (100 ng LPS/well) for 1 h at RT. Unbound peptides were removed by washing and THP-1 monocytes (1×10⁶/ml) were added to each well and incubated for 6 h at 37°C. The ability of released LL-37 to inhibit LPS-induced IL-8 production by THP-1 monocytes was determined by ELISA. Results are expressed as percentage of untreated control (100% = untreated CF sputum). *** <i>p</i><0.001.</p

LL-37 neutralises LPS from clinical strains of <i>P. aeruginosa</i> isolated from CF patients with severe airway disease.

<p>THP-1 monocytes were pre-treated with 5 µg/ml LL-37 for 1 h before cells were stimulated for 24 h with (<b>A</b>) SE4 and (<b>B</b>) SE22 LPS isolates from CF patients with severe airway disease (1, 10 and 100 ng/ml). IL-8 levels in cell-free culture supernatants were quantified by ELISA. ** <i>p</i><0.01; *** <i>p</i><0.001 compared to corresponding LPS alone (- LL-37). THP-1 monocytes were pre-treated for 1 h with LL-37 (5 µg/ml) and then washed with sterile PBS to remove extracellular, non-incorporated, exogenous LL-37. The cells were resuspended in fresh media and stimulated with 50 ng/ml (<b>C</b>) SE4 and (<b>D</b>) SE22 LPS for 24 h. IL-8 levels in cell-free culture supernatants were quantified by ELISA. Results are expressed as percentage of control response (100% = LPS alone). *** <i>p</i><0.001.</p