Ethics statement and isolation of human PMN.
Blood samples were obtained after informed consent from donors enrolled at the NIH Clinical Center under institutional review board-approved protocols 93-I-0119, 05-I-0213, or 99-CC-0168. Human polymorphonuclear leukocytes (PMN) were isolated from venous blood samples as previously described (20
). Sera were prepared using Becton Dickenson serum separator tubes according to manufacturer's instructions and stored on ice for immediate use or frozen at −80°C in single-use aliquots.
Bacterial strains and culture.
G. bethesdensis strain NIH1.1 (strain CGDNIH1T; ATCC BAA-1260) was grown to mid-log phase at 37°C in YPG medium (5 g yeast extract, 3 g peptone, and 10 g glucose per liter). Escherichia coli TOP10 and K1/r were grown in Luria-Bertani (LB) broth or Trypticase soy broth (TSB) at 37°C or as indicated. For each strain, A600 was used for enumeration of washed bacteria after appropriate conversion factors (no. of CFU/optical density [OD]) were determined.
Luminol-enhanced neutrophil chemiluminescence.
PMN were suspended at 1.1 × 106/ml in RPMI 1640 medium with 5.5% autologous serum, 27.5 mM HEPES (pH 7.4), and 55 μM luminol, and 90 μl was aliquoted into a 96-well polypropylene white plate. After a 30-min incubation period at 37°C in a 5% CO2 incubator, 10 μl of washed bacteria (multiplicities of infection [MOI] of 100:1, 10:1, 1:1) was suspended in saline and added to the plate. Saline alone was added as a control to some wells of the plate, and luminometry was performed in an Anthos Zenyth 3100 set at 37°C, with readings of 1 s per well taken every 2.4 min. Results are expressed kinetically or converted to area under the concentration-time curve (AUC) by the GraphPad Prism 5.0 software package.
Bacterial internalization assays.
Bacteria were labeled with pHrodo according to the manufacturer's instructions (Invitrogen, Carlsbad, CA) and kept at 4°C until use. For flow cytometry experiments, bacteria and PMN (MOI = 10 bacteria:1 PMN) were added to a 96-well plate with RPMI 1640 containing 25 mM HEPES (pH 7.2) and 10% autologous serum for a final volume of 200 μl. The plate was incubated at 37°C, and phagocytosis was stopped at each time point by addition of 50 μl of 10% neutralized buffered formaldehyde to each well. For the 0-min time point, formaldehyde was added to the wells immediately prior to addition of bacteria. After the final time point, the plate was stored at 4°C and read within 24 h on a FACSCalibur flow cytometer (BD Biosciences). Cells were gated by forward scatter and side scatter to exclude debris and extracellular bacteria. The pHrodo signal was measured in the FL2 channel, and the pHrodo+ threshold was set based on the 0-min time point and applied to all samples. For microscopy studies, EtOH-washed coverslips were placed in each well of a 24-well culture plate (Corning, NY) and dried. Assays were performed in RPMI 1640 containing 25 mM HEPES (pH 7.2), and the indicated numbers of PMN with or without additives were added. After thorough mixing, plates were centrifuged at 500 × g and placed in a humidified incubator at 5% CO2 and 37°C. At the indicated times, plates were centrifuged as indicated above, and the supernatant was aspirated, replaced with buffered formalin fixative, and placed at 4°C for at least 30 min. Coverslips were then mounted and observed using a Leica AF 6000 LX fluorescence microscope. A minimum of 10 fields (>100 cells) were imaged, and the numbers of PMN per field and the numbers of G. bethesdensis organisms in each PMN were enumerated in a blinded fashion.
Complement binding studies.
Saline-washed bacteria (2 × 108) were suspended in 1 ml RPMI and incubated at 37°C with the supplements and durations indicated in the figure legends. After 3 washes in 1 ml ice-cold PBS each, the bacteria were boiled in SDS-PAGE sample buffer with reducing agent and then subjected to Western blotting using standard methods. C3 and C3-deficient sera and goat anti-human C3 and C9 antisera were obtained from Complement Technology, Inc. (Tyler, TX). Primary antibodies were used at 1:2,000 and detected with an anti-goat horseradish peroxidase (HRP) conjugate (Santa Cruz) and Pierce West Pico chemiluminescent detection reagents.
Neutrophil killing assays.
All assays were performed as previously described (14
Neutrophil apoptosis studies.
Apoptosis of normal PMN or CGD PMN (CGD PMN) was estimated by nuclear morphology, DNA fragmentation, or flow cytometry for active caspase-3. PMN were incubated in RPMI 1640 buffered with 10 mM HEPES containing 10% autologous human serum (where indicated) in the presence or absence of G. bethesdensis and/or 1 μg/ml anti-FAS antibody (clone CH-11; MBL) for the indicated times. Freshly isolated PMN were also assessed by these methods and typically displayed <5% apoptosis. For morphological analysis, cells were subjected to cytospin, stained with the HARLECO Hemacolor stain set (EMD Chemicals), and imaged, and at least 200 cells were scored as healthy (typical multilobed nuclei) or apoptotic (condensed nuclei or “ghost” cell membranes lacking nuclei). To assess DNA fragmentation, a modified terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay (APO-BRDU kit; BD Pharmingen) was used. Briefly, cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 15 min, washed with PBS, and stored in 70% ethanol at −20°C. For the staining procedure, control cells and experiment samples were washed with kit wash buffer, incubated with DNA labeling solution for 1.5 h at 37°C with agitation every 15 min, washed twice with kit rinse buffer, and incubated with antibody labeling solution for 30 min at room temperature (RT) in the dark before fluorescence-activated cell sorting (FACS) analysis (conducted within 3 h). All centrifugations were conducted at 400 × g for 3 min at 4°C. Active caspase-3 was measured after washing cells once in 1% heat-inactivated fetal calf serum (FCS) and 1% bovine serum albumin (BSA) in PBS and twice with permeabilization buffer (1% heat-inactivated FCS, 0.1% saponin in PBS), followed with incubation for 15 min on ice to complete permeabilization. Cells were then stained with anti-active caspase-3–phycoerythrin (PE) antibody (BD Biosciences) for 30 min on ice in the dark, washed with permeabilization buffer, and fixed with 2% paraformaldehyde before FACS analysis.
Antimicrobial activity assays.
Bacteria were grown to mid-logarithmic phase, washed in 150 mM NaCl, and enumerated as described above. Antimicrobial activity was determined in RPMI 1640 containing 10 mM HEPES (pH 7.4). A peptide corresponding to the mature human cathelicidin peptide (LL-37) was synthesized commercially (Biosynthesis, Inc., Lewisville, TX), dissolved in 20 mM sodium acetate buffer at pH 4.0, and stored at 4°C. Hydrogen peroxide was freshly diluted from a 30% stock solution. Growth inhibition of inocula of 106/ml was determined after the indicated time points by plating (using sterile beads to achieve a uniform distribution on each plate) saline dilutions of cultures on appropriate media (either YPG or LB agar) and is expressed as the number of CFU/ml or percent control (buffer alone, plated at indicated times). Data and statistical analyses were performed using Microsoft Excel and GraphPad Prism for Mac, version 5.0.