Hishita T, Tada-Oikawa S, Tohyama K, Miura Y, Nishihara T, Tohyama Y, Yoshida Y, Uchiyama T, Kawanishi S

Hishita T, Tada-Oikawa S, Tohyama K, Miura Y, Nishihara T, Tohyama Y, Yoshida Y, Uchiyama T, Kawanishi S. a neutrophil defect may contribute significantly to unresolved illness(7). The prominence of sepsis in neutropenic individuals(8) also shows both the part of the neutrophil in defence against this organism and the clinical importance of understanding how this pathogen subverts the innate immune response. generates highly diffusible harmful secondary metabolites known as phenazines, which are critical for virulence and cytotoxicity in and mouse illness models(9), and it is the only organism to produce a specific phenazine, named pyocyanin(10). We have demonstrated pyocyanin, at concentrations recognized in sputum of cystic fibrosis individuals (11), induces a rapid, serious, and selective acceleration of neutrophil apoptosis illness, mice infected having a pyocyanin-producing strain, as compared having a pyocyanin-deficient but normally genetically identical strain, also showed accelerated neutrophil apoptosis and impaired bacterial clearance(13). Neutrophils are short-lived cells. Two major pathways to apoptosis are recognised: one proceeds through death receptor signalling, via membrane-associated signalling complexes and caspase-8 activation, and a second stress pathway, known to be controlled by oxidant stress, is definitely mediated by mitochondria and controlled by bcl-2 family members(14). The mechanisms of pyocyanin-induced acceleration of neutrophil apoptosis are mainly unfamiliar but may involve ROI generation and modified redox status(12). It is also unclear why neutrophils are exquisitely sensitive to pyocyanin. We consequently investigated the mechanisms of pyocyanin-induced apoptosis in neutrophils, and describe a novel pathway of pathogen-mediated neutrophil apoptosis, characterised by lysosomal acidification and activation of cathepsin D (CTSD). Materials and Methods MP-A08 Neutrophil isolation and tradition Human neutrophils were isolated by dextran sedimentation and plasma-Percoll (Sigma, Poole, UK) gradient centrifugation from whole blood of normal volunteers(15). The studies were authorized by the South Sheffield Study Ethics Committee and subjects offered written, educated consent. Purity of neutrophil populations ( 95%) was assessed by counting 500 cells on duplicate cytospins. Neutrophils were suspended at 2.5 106/ml in RPMI with 1% penicillin/streptomycin and 10% FCS (all Invitrogen, Paisley, UK) and cultured in 96 well Flexiwell plates (BD Pharmingen, Oxford, UK). Preparation and analysis of pyocyanin Pyocyanin was prepared by photolysis of phenazine methosulphate (Sigma) and purified and characterised as previously explained(16). Assessment of viability and apoptosis Nuclear morphology was assessed on Diff-Quik-stained cytospins, with blinded observers counting 300 cells per slip on duplicate cy tospins. Necrosis was assessed by trypan blue exclusion and was 2% unless indicated. On the other hand, neutrophils were washed in PBS and stained with PE-labelled Annexin V (BD Biosciences, San Jose, CA) and TOPRO?-3 iodide (Molecular Probes, Paisley, UK) to identify apoptotic (Annexin V+) and necrotic (TOPRO-3+) cells(17). Samples were analyzed using a FACSCalibur flow cytometer (BD Biosciences). Twenty MP-A08 thousand events were recorded and data was analyzed by CellQuest software (BD Biosciences). Caspase-3 activity assay Caspase 3 activity was determined by measuring enzymatically cleaved fluorescent substrate (DEVD-AMC, Bachem, Weil am Rhein, Germany) as previously described(18). Neutrophil lysates were prepared by re-suspension of treated cells in lysis buffer (100 mM HEPES, pH 7.5, 10% MP-A08 w/v sucrose, 0.1% CHAPS, 5 mM DTT) at a concentration of 1108/ml. Lysates were frozen at ?80C until required. Using the FLUSYS software package for the Perkin-Elmer LS-50B fluorimeter, lysate equivalents of 5 million neutrophils were co-incubated with 20 M Ac-DEVD-AMC in DMSO. Kinetic data was collected for at least 20 minutes to ensure stability of activity. A MP-A08 known amount of free AMC was used to calibrate the system and allowed calculation of caspase-3 activity. In separate experiments, executioner caspase (caspases 3 and 7) activity was measured using a Caspase-Glo 3/7 Assay (Promega, Madison, WI). Neutrophils were cultured at 5106/ml and treated with media (control), pyocyanin [50 M] and pyocyanin with dbcAMP (100 M) for 3 h. Cells were directly transferred to a white 96-well flat-bottomed plate (Dynex Technologies) at a density of 62,500 cells per well in a 25 l volume.) An equivalent volume of caspase-Glo 3/7 buffer mixed with substrate reagent was added to each well. The plate was read using a Lumistar Galaxy Luminometer (BMG Labtechnologies, Offenburg, Germany) at 25C for 200 cycles. ATP and glucose measurements ATP was measured using a commercially-available bioluminescent kit (Sigma) using a Lumistar Galaxy Luminometer. Glucose was assayed by detecting change MP-A08 in glucose concentration in lysates and culture supernatants using a commercial kit (Sigma), as previously described(19). Neutrophils were JV15-2 cultured in RPMI alone, with a glucose concentration of 2 mg/ml. Both assays were standardised using known concentrations of ATP and glucose respectively (data not shown). Modulation of pyocyanin-induced apoptosis Neutrophils were incubated in the presence and absence of pyocyanin following pre-incubation with candidate modulators of pyocyanin-induced apoptosis. Except where indicated, a concentration of.