DOES LINEZOLID MODULATE LUNG INNATE IMMUNITY IN A MURINE MODEL
OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS PNEUMONIA?
Morohunfolu E. Akinnusi, M.D.; Angela Hattemer, B.S.; Wei Gao; M.S.; Ali A. El-Solh, M.D.,
M.P.H.
MATERIALS AND METHODS
Organism and growth conditions
MRSA ATCC 33591 was obtained from the American Type Culture Collection
(Manassas, VA, USA). Bacteria was grown from a frozen stock for 6 to 10 h in LB broth and
then diluted 1/100 into fresh LB broth (4:1 flask-to-medium ratio) and grown for an additional 16
to 18 h with shaking (180 rpm). Stationary-phase bacteria were harvested by centrifugation at
room temperature, washed twice with endotoxin-free phosphate-buffered saline (PBS)
(Mediatech, Herndon, VA), and resuspended in endotoxin-free PBS to the desired concentration
as estimated by optical density and confirmed by quantitative plate counting. Minimum
inhibitory concentration (MIC) values were determined by the broth microdilution methodology
according to the Clinical and Laboratory Standards Institute (CLSI) (15).
Mouse model of pneumonia
Animal guidelines were followed in accordance with the Institutional Animal Care and
Research Advisory Committee and the animal protocols followed in this study were approved by
the local Animal Use and Care Committee. Specific-pathogen-free two-month-old female
BALB/c mice were purchased from the Charles River Labs (Troy, NY). The mice were housed in
the animal care facility of our institution in filter-top cages and allowed to acclimate to their new
environment for 1 week and received identical daily care for the duration of the experiment. To
determine the dose at which S. aureus would replicate in the lungs, 6 mice each were inoculated
with 3 x 108, 9 x 108, or 2x109 CFU ATCC 33591 and monitored at least twice daily. The
inoculums were deposited into the trachea with a 26 gauge needle with the animal in a 60 degree
incline position. Following inoculation, the animals were allowed to recover in an atmospherecontrolled chamber with the appropriate O2 concentration.
At 24h, 48h, and 72h after
inoculation, or when mice reached a moribund state, defined by hunched posture, piloerection,
labored breathing, immobility, and loss of resistance to handling, mice were euthanized by
intraperitoneal injection of an overdose of pentobarbital. Both lungs were harvested and
homogenized for quantitative culture as described previously (16).
Linezolid treatment and sample collection
Linezolid (Pfizer Pharmaceuticals, New London, CT) was reconstituted with sterile 0.9%
sodium chloride. Starting 12 hours post inoculation, mice were treated with either linezolid at a
dosage of 80 mg/kg of body weight every twelve hours intravenously (i.v.), vancomycin 110
mg/kg every 12 hours (i.v.), or no drug (control group). These doses in mice were calculated to
simulate human therapeutic exposures [based on area under the 0–24 h concentration–time curve
(AUC0–24) or t > MIC] using mouse pharmacokinetic data for vancomycin (17) and linezolid
(18). At 24 h, 48 h, and 72 h after inoculation, the animals were sacrificed and the appropriate
tissues harvested. The heart and lungs were then removed en bloc and the lung vasculature
cleared by injecting 2 ml saline into the right ventricle. For bronchoalveolar lavage (BAL), the
lungs were lavaged with 5 x 1 ml normal saline via a 25 gauge-catheter inserted in the trachea.
Aliquots of BAL fluid recovered from individual animals were pooled and stored at –80°C for
cytokine analysis. The thorax was opened, the trachea was visualized and cannulated with a 25gauge catheter, the left hilum was sutured, and the left lung was removed and homogenized in
2ml of sterile H2O with protease inhibitor (Complete, Roche Applied Science; Indianapolis, IN,
USA). The right lung was lavaged with five separate 1ml aliquots of 0.9% NaCl/0.6mM EDTA
at 370C and then fixed by intratracheal instillation of 4% paraformaldehyde at a transpulmonary
pressure of 15 cmH2O. Samples were obtained from a total of 6 mice per treatment group (three
treatment groups: mice treated with linezolid, mice treated with vancomycin, and mice treated
with placebo) at each time point from repeated identical experiments.
Culture
Serial dilutions of lung homogenates were plated onto trypsin-soy agar plates containing
10µg/ml of ampicillin, incubated overnight at 37o C, and read the following day to obtain the
numbers of CFU.
Cell counts
The percentage of neutrophils infiltrating the lungs of infected mice was determined by
modified Wright’s staining of leukocytes isolated from enzymatically digested tissue. Briefly,
lungs were incubated for 60 min at 37°C with digestion buffer (PBS containing 0.1% BSA, 0.01
M MgCl2, and 7 mM NaN3) to which 250 µg/ml DNase I and 3.3 mg/ml collagenase A was
added. Cell suspensions were passed through a 70-µm nylon cell strainer (BD Biosciences) and
cytospun onto polylysine-coated slides. Slides were prepared using a standard, modified Wright’s
staining protocol and leukocytes were enumerated by light microscopy.
Measurements of BAL cytokines and metalloproteinase-9
BAL fluid was assayed for the presence of pro- and anti-inflammatory cytokines using
Cytometric Bead Array flex sets (BD Pharmingen) which allow for simultaneous measurement
of MCP-5 (analogous to MACP-1 in humans) and IL-6 levels. The concentration of MMP-9, in
BAL supernatants was determined by specific enzyme-linked immunosorbant assays (R&D
Systems, Minneapolis, MN) according to the manufacturer's instructions.
MMP-9 zymography
Gelatinase activity was assayed by the method of Hibbs et al. (19). Briefly, a 30-µl
aliquot of each BAL sample was incubated with 1 ml of sterile H2O and 100 µl of gelatinagarose beads (Sigma-Aldrich) overnight at 4°C to enrich for MMPs. Beads then were
centrifuged at 10,000 x g for 1 min, resuspended in nonreducing Laemmli sample buffer (lacking
2-ME) and incubated for 30 min at room temperature (samples are not boiled before loading).
After centrifugation at 14,000 x g for 2 min, equal volumes of the eluate were resolved on a 7.5%
nonreducing
SDS-PAGE
containing
4
mg/ml
gelatin
(Sigma-Aldrich).
Following
electrophoresis, gels were washed three times with 50 mM Tris-HCl (pH 7.5) containing 2.5%
Triton X-100, 5 mM CaCl2, and 1 µM ZnCl2 and subsequently incubated for 24 h at 37°C in the
same buffer containing 1% Triton X-100. Gelatin activity was visualized by staining gels with
0.5% Coomassie blue and destaining with methanol/acetic acid.
Neutrophil apoptosis
Neutrophil apoptosis in BAL was assessed by annexin V and 7-aminoactinomycin D (7-AAD)
staining using an annexin V-PE kit (BD PharMingen, San Diego, CA) according to the
manufacturer’s instructions. A total of 1 x 106 cells recovered from BAL were washed,
preincubated in 10 µl of binding buffer containing 10 µg of mouse IgG, washed, then
resuspended in 100 µl of PBS containing 0.5 µg of FITC-1A8 or isotype control (BD
Pharmingen), and incubated for 15 min at 4°C. Cells were then washed and incubated with a 1/20
dilution of annexin V-PE in annexin V-binding buffer (BD Pharmingen) for 30 min at 4°C. As a
negative control, a parallel aliquot of cells were stained in the presence of 10 mM EDTA.
Following a final wash, the cells were resuspended in 400 µl of binding buffer containing a
1/10,000 dilution of the vital dye 7-AAD. Cells were analyzed on a dual-laser FACSCalibur flow
cytometer (BD Pharmingen), using FL1 for 1A8, FL2 for annexin V-PE, and FL4 for 7-ADD.
Ten thousand events were recorded and analyzed using CellQuest software (BD Pharmingen).
The neutrophil population were identified as 1A8-positive, early apoptotic cells as annexin Vpositive/7-ADD negative, and late apoptotic cells as annexin V-positive/7-ADD -positive. These
two populations were combined to give the total number of apoptotic cells.
Determination of MPO activity
Because neutrophil mediated inflammation may be overestimated in examination of lung tissues,
we have determined myeloperoxidase (MPO) enzyme activity in cell-free BAL fluid according
to a previously described method (20), with minor modifications. Aliquots of 50 µl of cell-free
BALF were mixed in microtiter plates with 200 µl of O-dianisidine dihydrochloride (1.25mg/ml
in PBS) plus BSA (0.1% wt/vol) containing H2O2 (0.05% = 0.4 mM). The MPO activities were
expressed as changes in absorbance at 450 nm.
Phagocytosis of apoptotic neutrophils
Alveolar macrophages were obtained by BAL as previously reported with minor modifications
(21) after 48 h of treating uninfected mice with either vancomycin (110 mg/kg every 12 hours) or
linezolid (80 mg/kg of body weight every 12 hours). Isolated AMs were cultured at 1.5 × 105
cells/ well in a Lab-Tek chamber slide system (chamber mounted on glass slide with cover;
Nalge Nunc International, Naperville, IL) for 2 d. Fresh neutrophils were recovered from
peritoneal cavity after an intraperitoneal injection of casein according to the modified method of
Van Epps and Garcia (22). In brief, mice were injected with 3.5 ml of a 2% saturated solution of
casein (Sigma Chemical Co., St. Louis, MO). After 15 h, mice were anesthetized and peritoneal
cells were recovered by lavage with 10 ml of cold (4°C) PBS. To minimize cell clumping, the
lavaged cells were washed and centrifuged at 4°C and visible cell clumps were removed by
adherence to glass pipettes. The washed peritoneal cells were further purified by centrifugation
through mono-poly resolving medium (MP Biomedicals; Solon, OH). The final pellet consisted
of 95% neutrophils, as evidenced by Diff-Quik staining.
Apoptosis of neutrophils was then induced by heating to 43°C for 45 min, followed by
incubation at 37°C in 5% CO2 for 3 h. This methodology yielded populations that included 33%
cells positively staining with annexin V with <10% positive for propidium iodide. Subsequently,
3 × 105 cells of apoptotic neutrophils were placed on the cultured AMs in triplicate and incubated
for 50 min at 37°C in 5% CO2/95% air. In the Lab-Tek chamber slide, noningested neutrophils
were removed by washing twice in PBS and then, to visualize internalized neutrophils, an MPO
stain was used. The percentage of phagocytosing macrophages was calculated as described
previously (23).